To test for subclinical disease in Q227X-inoculated tg66 mice and to confirm the transmission of the Y226X and G131V mutants to tg66 mice, we performed a second passage into tg66 mice. For each original mutant, brain homogenates from three first pass tg66 mice were intracerebrally inoculated into groups of 9–12 recipient tg66 mice. To date, one donor mouse (Y226X #B322–1) has caused clear clinical prion disease in 100% of challenged mice with an incubation period range of 167–185 dpi. Compared to the first passage, this incubation period is reduced by over 400 days, suggesting a rapid adaptation to tg66 mice and/or that the initial tg66 mice were inoculated with a very low titer. The other groups of experimental mice range from 40 to 225 days post-inoculation. Given the extended incubation periods observed on first passage final conclusions cannot be drawn at this time, and may require an additional two years of observation.