Metabolism of lanosterol (LAN), 24-methylene-24,25-dihydrolanosterol (24-methyleneDHL), dihydrolanosterol (DHL) and obtusifoliol (OBT) by purified human, plant (Sorghum bicolor) and fungal (Candida albicans) sterol 14alpha-demethylase (CYP51; P450(14DM)) reconstituted with NADPH cytochrome P450 reductases was studied in order to elucidate the substrate specificity and sterol stereo- and regio-structural requirements for optimal CYP51 activity. Both human and C. albicans CYP51 could catalyse 14alpha-demethylation of each substrate with varying levels of activity, but having slightly higher activity for their respective endogenous substrates in vivo, dihydrolanosterol for human CYP51 (Vmax = 0.5 nmol/min/nmol CYP51) and 24-methylene-24,25-dihydrolanosterol for C. albicans CYP51 (Vmax = 0.3 nmol/min/nmol CYP51). In contrast, S. bicolor CYP51 showed strict substrate specificity and selectivity towards its own endogenous substrate, obtusifoliol (Vmax = 5.5 nmol/min/nmol CYP51) and was inactive towards 14alpha-demethylation of lanosterol, 24-methylene-24,25-dihydrolanosterol and dihydrolanosterol. These findings confirm that the presence of the 4beta-methyl group in the sterol molecule renders the plant CYP51 incapable of 14alpha-demethylation thus revealing the strict active site conservation of plant CYP51 during evolution.