The contributions of cytochrome P450 3A5 to the metabolic clearance of marketed drugs is unclear, but its probable role is to augment the metabolism of several drugs that are largely cleared by P450 3A4. Selective metabolism by 3A4 is often a concern in drug development owing to potential drug-drug interactions and the variability of 3A4 and 3A5 expression. The contribution of P450 3A5 to these clearance pathways varies between individuals owing to genetic differences and similarities and differences in the metabolic properties of 3A5 compared with 3A4. To better understand the structural differences between P450s 3A4 and 3A5, the structure of 3A5 complexed with ritonavir was determined by X-ray crystallography to a limiting resolution of 2.91 Å. The secondary and tertiary structures of 3A5 and 3A4 are similar, but the architectures of their active sites differ. The 3A5 active site is taller and narrower than that of 3A4. As a result, ritonavir adopts a distinctly different conformation to fit into the cavity of 3A5 than seen for 3A4. These structural changes reflect amino acid differences that alter the conformation of the helix F through helix G region in the upper portion of the cavity and ionic interactions between residues in the beta-sheet domain that reduce the width of the cavity. The structural differences exhibited by 3A4 and 3A5 suggest that the overlap of catalytic activities may reflect molecular flexibility that determines how alternative conformers fit into the different active site architectures of the two enzymes.