The wooden panelling, ceiling beams and fireplace from the Oak Parlour at Gwydir Castle, near Llanrwst, were sold to an American newspaper owner in 1921.
They were shipped to New York but have not been seen since the 1930s.
It is now hoped they can be retraced and brought back to Wales.
Judy Corbett, who owns Gwydir Castle with her husband, Peter Welford, said it was bought by William Randolph Hearst, an American newspaper tycoon in 1921.
The 16th Century linen-fold panelling, the fireplace overmantel and the carved and moulded ceiling beams were dismantled, crated up and sent to America.
"He ended up assembling it in his billiard room in his apartment in New York," she said.
The apartment was known as the Clarendon, said to be the largest apartment in the world.
It was partly demolished in the 1930s, but the couple know the panelled rooms were taken out and stored.
"This is where we lose track of it," they said.
"Where did our room go?
"Did the Hearst family hold on to it?  Was it sold? Was it donated to an American museum?
"We have spent years looking through archives and records but can find no trace of it anywhere."
Numerous items of furniture, along with another whole room, were also sold off in 1921.
The panelling from the dining room was also bought by Mr Hearst.
But it was later discovered in a warehouse belonging to the Metropolitan Museum in New York, and was brought back to Wales and reinstalled at Gwydir in 1996.
Ms Corbett added: "The story of the dining room shows that miracles can happen. We would love to find our second missing room."

Carl Marston began writing about Colchester United for the East Anglian Daily Times on 21 November 1992 for a Third Division match at home to Rochdale.
The latest game is at Rochdale on Saturday - this time in League One.
Mr Marston, 48, said: "I'd like to keep doing it because, as a small club, you are closer to the manager and players."
The reporter joined the paper in 1989 and mainly covered non-league football before getting assigned to the Essex club.
The first game was a 4-4 draw - which ranks as the third favourite Colchester game he covered.
"You could say it was a baptism of fire, because the press box was new to me and I was hoping for an easy match, but it was changing constantly," Mr Marston said.
"Generally, access to the club is great and you wouldn't get that at a much bigger club.
"Once when I travelled on the team coach to Torquay, I'd finished filing my report only to find the bus had left.
"The manager George Burley realised and got them to turn back 30 miles - that's the sort of club it is."
While the club is still arguably best known for its shock 1971 FA Cup victory over Leeds United, Mr Marston has seen Colchester spend two seasons in the Championship and lose to Chelsea in the FA Cup.
Mr Marston said his personal high would be the 1998 play-off final win against Torquay at Wembley which saw Colchester promoted to League Division Two.
He was also delighted to see Colchester draw 0-0 at Yeovil to gain automatic promotion to the Championship in 2006, because he was due to get married on the day of the play-off final.
One of the other biggest changes has been the club's move from Layer Road to the Weston Homes Community Stadium in 2008.
Mr Marston said: "Layer Road has a lot of memories, but the facilities were very basic - the press box windows used to steam up and half-time tea was served in the laundry room - but it certainly had character.
"The new ground is fantastic and has potential if they can get into the Championship again, but it is soulless compared to Layer Road."
The owners of a 16th Century Tudor mansion in Conwy county are appealing to people in the United States to help them find a missing room.
[NEXT_CONCEPT]
A football reporter is about to reach the landmark of filing his thousandth match report covering the same club.