The i.p. administration of pentobarbital using an escalating drug-dose schedule for an 11-day period resulted in the establishment of dependence on pentobarbital in male rats. Mean plasma pentobarbital levels were approximately 5 micrograms/ml during the first 3 days of the infusion period. Subsequently, there was observed a dose-responsive increase in plasma pentobarbital levels for the next 5 days, with a decline in pentobarbital levels noted during the final 3 days of the pentobarbital infusion period. Removal of pentobarbital from the infusate resulted in a rapid decline in plasma levels to less than 50% by 8 hr into the drug-free period and to barely detectable levels by 24 hr. This was correlated with a steadily increasing occurrence of withdrawal signs, with a peak occurrence by 7 to 9 hr after initiation of the drug-free period. Spontaneous locomotor activity was significantly greater in pentobarbital-dosed animals during withdrawal than in saline-infused control rats. The i.p. infusion of pentobarbital is a quick and reliable method for the study of barbiturate dependence in the rat.