Cytochrome P450 2D6 (CYP2D6) catalyzes the oxidation of substrates with a positively charged nitrogen atom 5-7 angstroms from the site of the oxidation. The active-site topology of CYP2D6 is examined here with phenyl-, 2-naphthyl-, and p-biphenyldiazene, which react with P450 enzymes to form sigma-bonded aryl-iron (Fe-Ar) complexes. Ferricyanide-mediated migration of the aryl group from the iron to the porphyrin nitrogens produces the N-arylprotoporphyrin IX regioisomers (NB:NA:NC:ND, in which the aryl group is bound to the nitrogen of pyrrole rings B, A, C, and D, respectively) in the following ratios (zero means <5%): phenyl, 10:90:00:00; 2-naphthyl, 09:91:00:00; and p-biphenyl, 16:84:00:00. These results suggest that the CYP2D6 active site is open above pyrrole ring A and to a small extent above pyrrole ring B but is closed above pyrrole rings C and D. This geometry differs from those determined by the same method for P450s for which crystal structures are available. Replacement of Asp-301 by a Glu, which preserves the carboxylate side chain, causes no detectable change in the N-aryl porphyrin regioisomer patterns and only minor changes in the catalytic activity. Replacement of Asp-301 by an Asn or Gly, which eliminates the negatively charged side chain, suppresses migration of the aryl groups to pyrrole ring B without impairing migration to pyrrole ring A and virtually abolishes catalytic activity. These results provide a refined model of the active site of CYP2D6. They confirm, furthermore, that the loss of activity observed when Asp-301 is replaced by a neutral residue is due to loss of the charge-pairing interaction with the substrate positive charge and/or subtle structural effects in the vicinity of pyrrole ring B, but not to major structural reorganization of the active site.