Binding of substrates to the active site of cytochrome P450 enzymes largely relies on hydrophobic interactions. However, other binding interactions can take place giving the enzyme high regioselectivity and even stereoselectivity. For instance, within the major human cytochrome P450s involved in drug metabolism, cytochrome P4502D6 (CYP2D6) relies on an ion-pair interaction as a major binding factor. There are now a number of substrates reported that have routes of metabolism ascribed specifically to cytochrome P4502C9 (CYP2C9), the isoform mainly responsible for tolbutamide hydroxylation. Although chemically diverse, these substrates have the capability to be hydrogen bond donors (or acceptors). The substrate specificity has been rationalized in terms of a hydrogen bond donor/acceptor model and, by use of molecular modeling, an active site template model for CYP2C9 has been generated. The substrates modeled were phenytoin, warfarin, ibuprofen, naproxen, diclofenac, delta 1-tetrahydrocannabinol, 58C80, and tolbutamide. In addition to the substrates, the potent, selective inhibitor sulfaphenazole was also included in the modeling. An initial hydrogen bond donor site (N2) was identified on phenytoin, the most rigid of the substrates. Corresponding hydrogen bond donation sites were then identified on all of the molecules studied. Using molecular modeling, the site of metabolism and the hydrogen bond donation sites of the molecules were then overlaid on phenytoin to produce the putative active site model. The resultant model is described by a, the distance between the site of metabolism (Y), and the hydrogen bond donor heteroatom (X) and C, the angle between this and the hydrogen bond. The mean dimensions (+/- SD) for the nine substrates and one inhibitor (a = 6.7 +/- 1.0 A, C = 133 +/- 21 degrees) illustrate the degree of overlap achieved.