Background The mechanisms of action of volatile anesthetics are still unknown. Recently, the use of genetics as a means to investigate anesthetic action has increased in scale. However, only limited forward genetic approach studies were performed in mammals, especially with volatile anesthetics as the selection agent. In the present study, a selective breeding process was designed to produce strains of mice with different sensitivity to isoflurane. Methods One hundred and sixty male and female virgin outbred ICR/CD-1 mice at 65-70 days of age were selected as original generation, and the median effective dose (ED50) of inhaled isoflurane were measured by probit analysis with the loss of righting reflex as the endpoint of anesthesia. The most sensitive males and females were selected and mated one another randomly, as with the most resistant males and females. Thus two branches of mice (sensitive and resistant to isoflurane) were created and allowed to produce the next generation. At 65-70 days of age, screening experiment was performed in offspring, by selecting the most sensitive mice in sensitive branch and the most resistant mice in resistant branch. Selected males and females within each branch were mated one another randomly to produce the following generation. The same procedure was performed in the offspring. The process of screening and breeding was repeated for 8 generations, and then strains were conserved by mating the offspring one another randomly within each branch for 3 generations. Each pair of mice was allowed to produce the second litters as a backup, and isoflurane ED50 was measured in mice from the second litters. Results lsoflurane righting reflex ED(50)s (95% confidence limit (CL)) in original mice were 0.65% (0.58%-0.72%) in females and 0.63% (0.56%-0.69%) in males. After the 4th generation, isoflurane ED(50)s in resistant branch were significantly higher than those in sensitive branch (P <0.05), for both in females and males. In the 11th generation, isoflurane ED50 in the two branches differed by 32% in females and 36% in males. Conclusions After 8 generations of selective breeding and 3 generations of strain conservation, two strains of mice with high and low sensitivity to isoflurane were developed. The separation of inhaled anesthetic requirement in parents could be transferred to the offspring in mice. Chin Med J 2010;123(10):1315-1319