The combined effect of time and temperature on the stability of two avian influenza virus (AIV) isolates concentrated with polyethylene glycol (PEG), stored at different temperatures, and used in the preparation of avian influenza vaccine was evaluated in turkeys at 24 hr and at 12, 24, 30, 36, and 42 months of storage. The differences detected between antibodies raised in turkeys by vaccines made from isolates under different storage conditions, times, and temperatures were not significant (P > 0.05), especially with vaccines prepared from one isolate. Virus recovery rates following challenge studies of vaccinated birds were similar. However, birds that were vaccinated twice had lower rates of virus recovery from the trachea, lungs, pancreas, and fecal samples following challenge infection. The results suggest that if stable isolates of AIV can be identified, such isolates can be rapidly concentrated with PEG and stored at -20 C or -196 C for at least 42 months without any loss of potency in the vaccine prepared from these isolates. This would reduce the costs associated with vaccine storage and subsequent expiration dates.