Five N-monosubstituted chlorofluoroacetamides have been tested as potential specific irreversible inhibitors of the major phenobarbital-inducible form of rat liver cytochrome P-450 (P450IIB1). In vitro, N-(2-phenethyl)chlorofluoroacetamide was ineffective in causing a time-dependent loss of P450IIB1-mediated androstenedione 16 beta-hydroxylase activity in liver microsomes from phenobarbital-treated rats. However, addition of a nitro or bromo substitutent at the para position of the phenyl group or addition of a second phenyl group at the 1- or 2-position on the phenethyl side chain yielded compounds that caused a selective time-dependent decrease in androstenedione 16 beta-hydroxylase activity relative to four other P-450 form-specific androstenedione or progesterone hydroxylase activities monitored. The two compounds that were the most effective in inactivating P450IIB1 in vitro, N-(2-p-bromophenethyl) and N-(2-p-nitrophenethyl)chlorofluoroacetamide were also administered ip to phenobarbital-treated rats, and inhibition of cytochromes P-450 was assessed by in vitro assays of steroid and R- and S-warfarin hydroxylation in subsequently prepared hepatic microsomes. Both compounds selectively inhibited P450IIB1, and at a dose (200 mg/kg) of N-(2-p-nitrophenethyl)chlorofluoroacetamide that reduced androstenedione 16 beta-hydroxylase activity to approximately one-third of the control level, only two other activities, both attributable to P450IIB1, were decreased. In contrast, steroid and warfarin hydroxylase activities indicative of at least five other cytochromes P-450 were unaffected by the compound. These results indicate the feasibility of an empirical approach to the design of specific cytochrome P-450 inactivators.