As an approach to investigating mechanisms of anesthetic action, studies using selective breeding of animal stocks with different anesthetic sensitivity have increased during recent years. Mice are an ideal model for such studies due to their small size, short reproductive cycle, well-established behavioral endpoints of anesthesia, and well-known genetic background. Because single litters are not large enough for simultaneous selective breeding and conservation of the stock, mice must be used from successive litters. However, the stability of isoflurane anesthetic potency across successive litters has not been reported. In the present study, 24 (12 male and 12 female) outbred ICR mice were mated. Each pair was allowed to produce 2 successive litters. Offspring were separated by sex after weaning at 21 d of age. Reproductive characteristics were documented, including litter size at birth, sex ratio at weaning, and neonatal mortality. At 65 to 75 d of age, the median effective dose (ED50) of inhaled isoflurane was measured in mice from the 2 parities by using a bracketing design. Loss of righting reflex was chosen as the criterion for successful anesthesia. The 2 parities did not differ significantly with regard to reproductive parameters and isoflurane ED50 of offspring. These results indicate that offspring in the second litter from the same parents can be used for stock conservation and anesthesia research.