Reduction of serum cholesterol in man, particularly that carried in low-density lipoproteins, has been shown to reduce the incidence of coronary heart disease. Lanosterol 14a-demethylase (P-45014DM) catalyses the first step in the conversion of lanosterol to cholesterol in mammals and provides a possible intervention point for inhibiting cholesterol biosynthesis. While the related enzyme in fungi has been extensively studied (with antifungal azoles blocking this enzyme), ketoconazole, an orally active antifungal, inhibits rat 14-DM and lowers serum cholesterol in man but is relatively nonselective, inhibiting other cytochromes P-450 involved in steroid biosynthesis, cholesterol metabolism, and xenobiotic metabolism. Taking work on antifungals as a starting point, we undertook a search for azole inhibitors of mammalian 14-DM having minimal effects on other important P-450 enzymes. We report here the preparation and biological activity of the first non-steroidal selective inhibitor of mammalian lanosterol 14a-demethylase, compound 1 (RS-21607), as a potential strategy for cholesterol lowering in man. Compound 1 competitively inhibited lanosterol 14a-demethylation in hepatic microsomes from rat, hamster, and human with apparent Ki values of 2.5 ± 1.5, 1.4, and 0.79 ± 0.35 nM, respectively, and showed good selectivity relative to other cytochrome P-450 enzymes involved in steroid biosynthesis and drug metabolism. It was approximately 400 times more effective than ketoconazole in inhibiting cholesterol biosynthesis from [2-¹⁴C]acetate in human fibroblasts (IC50 values: 0.09 vs 37 nM). In male Syrian hamsters, compound 1 was more effective than its stereoisomers in reducing serum cholesterol in vivo; its dihydrochloride salt (1-2HCl) was superior to lovastatin and ketoconazole after 14-day dosing, showed a favorable effect on lipoprotein profiles (increasing HDL/total cholesterol ratio and decreasing LDL), and is currently undergoing clinical development as a hypocholesterolemic agent.