Serum is often frozen and banked for analysis at a later date. This study assessed the stability of 17 analytes in rat serum during refrigeration at 4 degrees C and extended storage at -20 degrees C (frost-free and nonfrost-free freezers) and -70 degrees C. Samples were analyzed by using an automated dry-slide chemistry analyzer at time 0 and then stored as aliquots for analysis at time points including day 7, 30, 90, and 360. After 7 d of refrigeration, only creatine kinase activity had varied by more than 10% of the starting value. Freezing at -70 degrees C was clearly superior to -20 degrees C where changes were observed in CO(2) as early as day 30 and alanine aminotransferase as early as day 90. Samples stored in frost-free and nonfrost-free -20 degrees C freezers did not differ significantly through day 90. Factors such as storage time and temperature should be considered when designing any retrospective study.