The ability of S9 liver fractions from uninduced rats to activate isophosphamide (IP) and trophosphamide (TP) to metabolites mutagenic for bacteria was compared to that of S9 fractions prepared from rats pretreated in vivo with three inducers of hepatic monooxygenase. Pretreatment of rats with phenobarbital (PB) and Aroclor 1254 increased IP and TP mutagenic activation by S9 fractions as compared to control and 3-methylcholanthrene (3-MC)-induced rat liver S9. Furthermore, the effect of mixed-function oxidase inhibitors, such as alpha-naphthoflavone, metyrapone and SKF 525-A on S9-mediated mutagenic activation of IP and TP was investigated. The data obtained suggest the involvement of a PB-inducible form of cytochrome P-450 in the activation of IP and TP to mutagenic species.