Cholesterol biosynthesis was examined in rat hepatic subcellular preparations (S-10) incubated with [24-3H]-lanosterol in the presence of 12 synthetic lanosterol analogs with unnatural side chains, 20-iso-24-dihydrolanosterol, 25-hydroxy-24-dihydrolanosterol, 25-hydroxycholesterol and cyclolaudenol. Cholesterol biosynthesis from 18 .mu.M [24-3H]-lanosterol was inhibited by 40 .mu.M 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.