The CYP2C subfamily of human liver P450 isozymes is of major importance in drug metabolism. The most abundant 2C isozyme, CYP2C9, regioselectively hydroxylates a wide variety of substrates. A major obstacle to understanding this specificity in human CYP2C9 is the absence of a 3D structure. A 3D model of CYP2C9 was built, assessed, and used to characterize explicit enzyme-substrate complexes using methods previously developed in our laboratory. The 3D model was assessed by determining its stability to unconstrained molecular dynamics and by comparison of specific properties with those of known protein structures. The CYP2C9 model was then used to characterize explicit enzyme complexes with three structurally and chemically diverse substrates: (S)-naproxen, phenytoin, and progesterone. Each substrate was found to bind to the enzyme with a favorable interaction energy and to remain in the binding site during unconstrained molecular dynamics. Moreover, the mode of binding of each substrate led to calculated preferred hydroxylation sites consistent with experiment. Binding-site residues identified for the models included Arg 105 and Arg97 as key cationic residues, as well as Asn 202, Asp 293, Pro 101, Leu 102, Gly 296, and Phe 476. Site-specific mutations are proposed for further integrated computational and experimental study.