Effects of buthiobate (S-butyl S'-p-tert-butylbenzyl N-3-pyridyldithiocarbonimidate), a fungicide which inhibits lanosterol 14α-demethylation catalyzed by a cytochrome P-450 (P-450_14DM) of yeast, on cytochrome P-450 of rat liver microsomes were studied. Buthiobate bound to limited forms of cytochrome P-450 in the microsomes with three different K_d values, 0.38, 1.56 and 13.6 μM. Buthiobate inhibited lanosterol 14α-demethylase activity of the microsomes with a 50%-inhibition concentration of 0.4 μM. This concentration was comparable to the lowest K_d of buthiobate for the microsomal cytochrome P-450 and also to the 50%-inhibition concentration of the inhibitor for lanosterol 14α-demethylation by yeast P-450_14DM. Buthiobate partially inhibited 7-ethoxycoumalin O-deethylase activity of the microsomes but inhibited neither benzphetamine N-demethylation nor p-nitroanisole O-demethylation. These observations suggest that cytochrome P-450s catalyzing drug oxiations are rather insensitive to buthiobate. These observations indicate that buthiobate is an unique inhibitor for hepatic microsomal cytochrome P-450 system, which inhibits cholesterol biosynthesis effectively but causes a little effect on drug oxidations.