Cytochrome P450 2E1 (CYP2E1), an enzyme that is induced by ethanol, plays an important role in the metabolism of various toxic and carcinogenic substances, including dialkylnitrosamines and small halocarbons. Little information is available on the active site of the enzyme. We report here the formation of aryl-iron complexes (Fe-Ar) in the reactions of human CYP2E1 with phenyldiazene, (2-naphthyl)hydrazine, and p-biphenylhydrazine. Migration of the aryl group from the iron to the porphyrin nitrogens within the intact active site produces the four possible N-arylprotoporphyrin IX regioisomers in the following ratios (NB:NA:NC:ND, where the subscript indicates the pyrrole ring): phenyl, 03:34:02:61; 2-naphthyl, 02:18:03:77; p-biphenyl, 02:52:04:42. The results indicate that the active site of human CYP2E1 is sterically unhindered directly above the iron for a distance of 10 A. It appears, furthermore, that the active site cavity is relatively open above pyrrole rings A and D but is closed above pyrrole rings B and C, a topology similar to that deduced for the rat enzyme.