Lec9 Chinese hamster ovary cells were found previously to be defective in the synthesis of N-linked glycans. This appeared to be the result of a defect in the synthesis of oligosaccharide lipid and lipid phosphate (Rosenwald, Stanley, and Krag. 1989. Mol. Cell. Biol. 9: 914-924). In this study we analyzed the steady state levels of long-chain polyisoprenyl lipids in Lec9 cells. We found that Lec9 cells are defective in the synthesis of dolichol. They accumulated a presumed precursor to dolichol, cis-a-unsaturated polyprenol and used this lipid in the synthesis of oligosaccharide lipid. Chain lengths of the activated polyprenols in Lec9 were the same lengths as dolichols in parental cells. Lec9 cells had increased levels of monosaccharylphosphoryl lipid and decreased levels of oligosaccharylpyrophosphoryl lipid compared to parental cells. The defect in Lec9 cells was specific for dolichol synthesis, since other aspects of [3H]mevalonate metabolism in Lec9 cells were the same as in parental cells. We hypothesize that Lec9 cells are defective in polyprenol reductase activity.