Abstract Tolerance to pentobarbital developed 4 hr. after the first injection and reached a peak at 17–22 hr., after which it decreased to nonsignificant levels by 48 hr. The duration and frequency of administration of pentobarbital affected the degree of development of tolerance to pentobarbital. PTI (Percentage Tolerance Index) decreased progressively with the increase in the number of injections. When four injections were given within the span of 28 hr., the animals showed a greater degree of tolerance on the third injection which was administered 24 hr. after the initial injection. Tolerance was also present on the fourth injection but to a lesser degree when compared with the third injection. The experimental data from this study suggest that tolerance to pentobarbital does develop and is the result of the pentobarbital stimulating its own metabolizing enzyme.