We synthesized three water-soluble biotin-tagged compounds with different prenyl chain lengths, biotinylated farnesal (BF), biotinylated C₅₅ polyprenal (BP55), and biotinylated C₈₀-polyprenol (BP80), and examined their effects on in vitro dolichol synthesis from farnesyl diphosphate. BF and BP55 did not affect the dolichol synthesis, whereas BP80 inhibited the reduction pathway from polyprenol to dolichol, accompanied by a decrease in the entire polyprenol and dolichol synthesis. Comparison of BP80 with eighteen detergents, including Triton X-100, CHAPS, octylglucoside, deoxycholate, and Tween 80, revealed the specific effect of BP80 on the reduction pathway. On SDS-polyacrylamide gel electrophoresis, BP80 was detected in an associated form with a 50 kDa protein. These results suggest that the reduction of polyprenol to dolichol in the dolichol biosynthetic pathway proceeds with the recognition of the polyprenol chain length by a 50 kDa protein.