YM-16638 ([[5-[[3-(4-acetyl-3-hydroxy-2-propylphenoxy)propyl]thio]-1, 3, 4-thiadiazol-2-yl]thio] acetic acid) showed a strong hypocholesterolemic effect in humans and monkeys. To clarify the mechanism of this hypocholesterolemic effect, the action of YM-16638 on cholesterol biosynthesis in the cultured human hepatoma cell line HepG2 and cynomolgus monkey liver was examined. Cholesterol biosynthesis activity derived from [¹⁴C]acetic acid, [³H/¹⁴C]mevalonic acid or [¹⁴C]isopentenyl pyrophosphate substrates was significantly decreased, but not that from [³H]farnesyl pyrophosphate or [³H]squalene substrates in HepG2 cells treated with YM-16638. Simultaneously, treatment of these cells with YM-16638 changed neither the rate of apolipoprotein B synthesis from [³⁵S]methionine nor its secretion. In addition, the activities of hepatic cholesterol biosynthesis enzymes HMG-CoA reductase, mevalonate kinase (MK), isopentenyl pyrophosphate isomerase (IPPI), farnesyl pyrophosphate synthase (FPPS), squalene synthase and squalene epoxidase were measured in monkeys fed a diet supplemented with YM-16638. Among these enzymes, MK, IPPI and FPPS activities in the YM-16638-treated group significantly decreased by 38%, 56% and 30%, respectively, when compared to those from control animals receiving no drug treatment. These results indicate that YM-16638 has the characteristics of a cholesterol biosynthesis inhibitor.