Cholesterol biosynthesis was examined in rat hepatic subcellular preparations (S-10) incubated with [24-³H]-lanosterol in the presence of twelve synthetic lanosterol analogs with unnatural side chains (1-12), 20-iso-24-dihydrolanosterol, 25-hydroxy-24-dihydrolanosterol, 25-hydroxycholesterol and cyclolaudenol. Cholesterol biosynthesis from 18μM [24-³H]-lanosterol was inhibited by 40μM lanosterol analogs. Among the analogs studied, 27-nordihydrolanosterol was most active in depressing cholesterol biosynthesis from lanosterol. The structure-activity relationship of lanosterol analogs and related compounds is discussed.