The catalytic domain of cytochrome P450 is thought to contact the lipid core of the endoplasmic reticulum membrane based on antibody epitope accessibility, protease susceptibility, and hydrophobic surfaces present on P450 structures of solubilized forms of the proteins. Quenching by nitroxide spin label-modified phospholipids of the fluorescence of tryptophan residues substituted into cytochrome P450 2C2, modified to contain tryptophan only at position 120, was used to identify regions of P450 inserted into the lipid core and to estimate the depth of penetration. Consistent with the proposed models of cytochrome P450-membrane interaction, the fluorescence of tryptophans inserted at residues 36 and 69 in the two segments of P450 2C2 flanking the A-helix and at residue 380 in the beta2-2 strand was quenched by nitroxide spin labels on carbon 5 of the fatty acid tails of the phospholipids within the lipid bilayer. The fluorescence of tryptophan at 380 was also strongly quenched by a spin label on carbon 12 of the fatty acids suggesting it was deepest in the membrane. However, fluorescence of tryptophan substituted at residue 225 in the F-G loop, which was predicted to be in the lipid bilayer, was not quenched by the spin labels at carbons 5 and 12 of the fatty acids. The pattern of quenching of fluorescence for tryptophans at the other positions tested, 80, 189, 239, and 347, was similar to the parent protein indicating they were not inserted into the lipid bilayer as expected. The results are consistent with an orientation of cytochrome P450 2C2 in the membrane in which positions 36, 69, and 380 are inserted into the lipid bilayer and residues 80 and 225 are near or within the phospholipid headgroup region. In this orientation, the F-G loop, which contains residue 225, could form a dimerization interface as was observed in the P450 2C8 crystal structure (Schoch, G. A., et al. (2004) J. Biol. Chem. 279, 9497).