Male and female patas (Erythrocebus patas) and cynomolgus (Macaca fascicularis) monkeys were treated with phenobarbitone (PB) and examined for the induction of various cytochrome P450 (P450)-mediated drug metabolizing enzymes. Hydroxylation of testosterone at the 6β, 2β, and 15β positions, metabolites normally associated with CYP3A P450s increased 2− to 5-fold in PB-treated animals. Induction of this P450 family was confirmed by the use of polyclonal antisera directed against the human CYP3A enzymes which inhibited both induced and constitutive 6β – and 15β -hydroxylase activities in both species of monkeys. The enzymatic activities testosterone 16β -hydroxylation, pentoxyresorufinO-dealkylation [PROD], and benzyloxyresorufinO-dealkylation [BZROD] typically associated with the rodent CYP2B subfamily in rodents were also examined. Testosterone 16β -hydroxylation activity was highly induced up to 15-fold in both species of monkeys to maximal levels similar to those induced in PB-treated male rats. BZROD was similarly induced up to 10-fold in both species of monkeys, but the maximal levels of BZROD achieved were substantially lower than those obtained in PB-treated rats. Finally, PROD yielded an idiosyncratic response showing substantial induction (> 30-fold) in certain patas monkeys (4 out of 8) but minimal (< 5-fold) induction in the other patas monkeys (4 out of 8) or any of the cynomolgus monkeys (0 out of 8). Immunodetection of various cytochromes using polyclonal antisera directed against rat cytochromes confirmed the induction of CYP3A proteins as well as the induction of protein(s) immunologically cross-reactive with rat CYP2B P450s. BZROD, PROD, or testosterone 16β-hydroxylase activities, were not inhibited by antibody to CYP3A P450s. However, high concentrations of polyclonal antiserum directed against rat CYP2B inhibited all three activities in PB-induced patas monkeys. In contrast, this antiserum failed to inhibit the hydroxylation of testosterone at the 6β or 2β positions. Induction of the CYP2C subfamily was observed by immunochemical detection. Interestingly, induction of this subfamily appeared to be more pronounced in cynomolgus than in patas monkeys. Finally, we failed to observe significant sex-dependent differences in P450-mediated enzymatic activities in either control or induced monkeys. These results confirm the induction of a similar spectrum of P450 proteins by PB in non-human primates to that which has previously been observed in rodents.