A new method for experimentally analyzing the role of enzymes involved in metabolizing mutagenic, carcinogenic, or cytotoxic chemicals is described. Spodoptera fugiperda (SF-21) cells infected with recombinant baculoviruses are used for high level expression of one or more cloned enzymes. The ability of these enzymes to prevent or enhance the toxicity of drugs and xenobiotics is then measured in situ. Initial parameters for the system were developed and optimized using baculoviruses engineered for expression of the mouse soluble epoxide hydrolase (msEH, EC 3.3.2.3) or the rat cytochrome P4501A1. SF-21 cells expressing msEH were resistant to trans-stilbene oxide toxicity as well as several other toxic epoxides including: cis-stilbene oxide, 1,2,7,8-diepoxyoctane, allylbenzene oxide, and estragole oxide. The msEH markedly reduced DNA and protein adduct formation in SF-21 cells exposed to [3H]allylbenzene oxide or [3H]estragole oxide. On the other hand, 9,10-epoxyoctadecanoic acid and methyl 9,10-epoxyoctadecanoate were toxic only to cells expressing sEH, suggesting that the corresponding fatty acid diols were cytotoxic. This was confirmed by showing that chemically synthesized diols of these fatty acid epoxides were toxic to control SF-21 cells at the same concentration as were the epoxides to cells expressing sEH. A recombinant baculovirus containing a chimeric cDNA formed between the rat P4501A1 and the yeast NADPH-P450 reductase was also constructed and expressed in this system. A model compound, naphthalene, was toxic to SF-21 infected with the rat P4501A1/reductase chimeric co-infecting SF-21 cells with either a human or a rat microsomal EH virus along with P4501A1/reductase virus. These results demonstrate the usefulness of this new system for experimentally analyzing the role of enzymes hypothesized to metabolize endogenous and exogenous chemicals of human health concern.