Cytochromes P450 (P450s) constitute a large superfamily of heme-containing enzymes, capable of oxidizing and reducing a variety of substrates. Cytochrome P450 2D6 is a polymorphic member of the P450 superfamily and is absent in 5-9% of the Caucasian population as a result of a recessive inheritance of gene mutations. Recently, the importance of aspartic acid 301 (Asp301) for the catalytic activity of P450 2D6, as indicated by a preliminary homology model, was confirmed by site-directed mutagenesis experiments. In this study, the heme moiety and the I-helix containing Asp301 were incorporated into the previously derived substrate model for P450 2D6, in the spatial orientations as derived from a recently improved protein model for P450 2D6, thereby incorporating steric restrictions and orientational preferences into the substrate model. The direction of well-defined hydrogen bonds formed between Asp301 and basic nitrogen atoms of P450 2D6 substrates was incorporated into the substrate model as well. Also, the position(s) of the basic nitrogen atom(s) of the substrates was/were allowed more flexibility. This was established through the attachment of an aspartic acid residue (representing Asp301) to the (protonated) basic nitrogen atom(s) of the substrates and superimposing the C alpha- and C beta-atoms of this aspartic acid residue in the fitting procedure instead of the basic nitrogen atoms. A variety of 8 substrates of P450 2D6 (comprising 17 known P450 2D6 dependent metabolic pathways) has been incorporated successfully into this refined and more restrictive substrate model.