Mycobacterium avium complex (MAC) causes chronic lung disease in immunocompetent people and disseminated infection in patients with AIDS. MAC is intrinsically resistant to many conventional anti-mycobacterial agents, it develops drug resistance rapidly to macrolide antibiotics, and patients with MAC infection experience frequent relapses or the inability to completely eradicate the infection with current treatment. Treatment regimens are prolonged and complicated by drug toxicity or intolerances. We sought to identify biochemical pathways in MAC that can serve as targets for novel anti-mycobacterial treatment. The cytochrome P450 enzyme CYP51 catalyzes an essential early step in sterol metabolism, removing a methyl group from lanosterol in animals and fungi, or from obtusifoliol in plants. Azoles inhibit CYP51 function leading to an accumulation of methylated sterol precursors. This perturbation of normal sterol metabolism compromises cell membrane integrity, resulting in growth inhibition or cell death. We have cloned and characterized a CYP51 from MAC which functions as a lanosterol 14-alpha-demethylase. We show the direct interactions of azoles with purified MAC-CYP51 by absorbance and EPR spectroscopy and determine the MICs of econazole, ketoconazole, itraconazole, fluconazole and voriconazole against MAC. Further, we demonstrate that econazole has a MIC of 4 µg/ml and a MBC of 4 µg/ml while ketoconazole has a MIC of 8 µg/ml and a MBC of 16 µg/ml. Itraconazole, voriconazole, and fluconazole did not inhibit MAC growth to any significant extent.