Pretreatment with sodium phenobarbital induces hepatic microsomal enzymes which are responsible for the metabolic breakdown of a large number of endogenous and exogenous chemical compounds. A previous study [K. P. DuBois and F. K. Kinoshita, Proc. Soc. exp. Biol. Med. 129, 699 (1968)] reported that phenobarbital pretreatment reduced the toxicity of various organophosphorus anticholinesterases; however, the exact mechanism for the increased detoxification was not investigated. In this study, the effect of phenobarbital pretreatment on the toxicity of soman was investigated. Male mice were injected daily for 4 days with sodium phenobarbital (100 mg/kg, i.p.) and used in the various experiments 24 hr after the last injection. Phenobarbital pretreatment produced a significant increase in liver weight and decreased the sodium pentobarbital (75 mg/kg, i.p.) induced sleep-time to 41 min compared to 141 min in controls. The lethality of soman was reduced following phenobarbital pretreatment. In control mice, the soman 24 hr LD50 values (microgram/kg) were 130, 393 and 42 following s.c., i.p. and i.v. administration, respectively, whereas in phenobarbital-pretreated mice the soman 24 hr LD50 values (microgram/kg) were 261, 746 and 63 following s.c., i.p. and i.v. administration respectively. Acetylcholinesterase activity was increased in the plasma (90%) but not in brain or diaphragm following phenobarbital pretreatment. Liver somanase activity was not affected. Liver aliesterase and serum aliesterase were both increased significantly following phenobarbital pretreatment. An increase in the amount of nonspecific binding sites for soman (esterases in liver and plasma) and not an increase in the metabolism of soman in vivo probably accounts for the protection afforded by phenobarbital pretreatment in mice.