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A terse, three-line communiqué from the Elysée presidential palace, issued shortly after midnight (2300 GMT), announced his decision to step down. His decision takes effect from 1200 local time (1100 GMT).
The result of the vote was decisive: 52.87% of the electorate voted "no", with 47.13% supporting the president. Turnout was over 80%. The result of voting in overseas territories has yet to arrive, but will not affect the outcome.
His resignation has caused shockwaves around the world. General de Gaulle, 78, is a towering figure in post-war politics: the symbol of Free France during the Nazi occupation in the Second World War, he founded the Fifth Republic in 1958 when he became President of France.
He was a controversial figure at home and abroad, but nonetheless revolutionised French political institutions, and became France's most powerful head of state since Napoleon III.
The Prime Minister, Maurice Couve de Murville, spoke in a television broadcast of his "profound sadness" at the result. He said it was "an event the gravity of which will very quickly appear to all people in France and in the world".
Vote of confidence
Robert Poujade, secretary-general of the Gaullist Party, was one of many paying tribute to General de Gaulle.
"He who inspired our struggles of yesterday will continue to inspire our struggles of tomorrow," he said. "In this ordeal, as he taught us, we shall not flinch one moment."
The proposals for reform which brought down the president were aimed at modernising the structure of government. General de Gaulle saw the issue as so crucial that he tied his own future to that of the referendum result. It turned the campaign into a vote of confidence in the General himself.
All attention now turns to former Prime Minister Georges Pompidou, who has never denied his intention to stand as President if Mr de Gaulle resigned. Mr Pompidou, controversially dismissed by the General after the elections last year, has long been regarded as his obvious successor.
With declarations due within the next month, the race is on to see who can follow one of the greatest acts in the history of France.
General de Gaulle retired to his country retreat at Colombey-les-Deux-Eglises, in north-east France, to write his memoirs. He died from a heart attack on 9 November 1970.
Georges Pompidou was elected President of France on 15 June 1969, with 58.22% of the vote. The left wing fragmented during campaigning, and the result was widely predicted.
During his time in office, he weathered the major financial crisis that followed General de Gaulle's resignation, devaluing the franc in August 1969. He was highly regarded in international politics, but at home, he is sometimes referred to as "the forgotten President" of the Fifth Republic.
He died of cancer while still in office in 1974, and was succeeded by Valery Giscard d'Estaing.
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