Trichloroethylene (TCE) has been shown to be toxic to experimental animals and humans. TCE oxide is a reactive electrophile formed during TCE oxidation and rearranges to acylating intermediates [Cai, H., and Guengerich, F. P. (1999) J. Am. Chem. Soc. 121, 11656-11663], which may be related to the toxicity. Mice treated with TCE have been reported to contain N(6)-dichloroacetylLys residues in P450 2E1, as detected by immunochemical methods. TCE can be oxidized by both P450 2E1 and (rat) 2B1. In this work, direct reaction of TCE oxide with either human P450 2E1, P450 2B1, or NADPH-P450 reductase was shown to lead to enzyme inactivation, and no recovery of the activity of either enzyme occurred, consistent with the view of inactivation reactions with Lys groups and not hydroxyls or Cys. Furthermore, Lys adducts were detected in the reaction of TCE oxide with both P450 2E1 and NADPH-P450 reductase, with a larger amount of N(6)-formylLys observed compared to N(6)-dichloroacetylLys in both cases. Inactivation of P450 2E1 during NADPH-dependent TCE oxidation was not observed, compared to control experiments. However, inactivation of P450 2B1 during NADPH-dependent TCE oxidation was detected. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry of tryptic peptides indicated that the direct reaction of TCE oxide with human P450 2E1 resulted in the modification of peptides containing Lys87 (AVKEALLDYK), Lys251 (VKEHHQSLDPNCPR), and Lys487 (YKLCVIPR), with either a formyl or dichloroacetyl group attached. Lys87 and Lys487 of human P450 2E1 appear to be modified during the oxidation of TCE, using the same approach. The results are considered in the context of comparison of species and P450s.