Although there are currently three generations of antifungal azoles on the market, even the third-generation agents show undesirable interactions with human cytochrome P450 (P450) enzymes. CYP46A1 is a cholesterol-metabolizing P450 in the brain that tightly binds a number of structurally distinct azoles. Previously, we determined the crystal structures of CYP46A1 in complex with voriconazole and clotrimazole, and in the present work we cocrystallized the P450 with posaconazole at 2.5 Å resolution. This long antifungal drug coordinates the P450 heme iron with the nitrogen atom of its terminal azole ring and adopts a linear configuration occupying the whole length of the substrate access channel and extending beyond the protein surface. Numerous drug-protein interactions determine the submicromolar Kd of posaconazole for CYP46A1. We compared the crystal structure of posaconazole-bound CYP46A1 with those of the P450 in complex with other drugs, including the antifungal voriconazole and clotrimazole. We also analyzed the accommodation of posaconazole in the active site of the target enzymes, CYPs 51, from several pathogenic species. These and the solution studies with different marketed azoles, collectively, allowed us to identify the determinants of tight azole binding to CYP46A1 and generate an overall picture of azole binding to this important P450. The data obtained suggest that development of CYP51-specific antifungal agents will continue to be a challenge. Therefore, structural understanding of the azole binding not only to CYPs 51 from the pathogenic species but also to different human P450s is required to deal efficiently with this challenge.