The Lec35 mutation (previously designated PIR) of Chinese hamster ovary cells is a recessive mutation that affects the participation of mannose-P-dolichol (MPD) in dolichol-P-P-oligosaccharide biosynthesis in vivo, even though MPD and the respective MPD-dependent mannosyltransferases are present. The Lec35 phenotype can be partially corrected by disrupting Lec35 cells and performing the transferase reactions in vitro, suggesting that the defect may be related to mislocalization of MPD. In this study, we examined the effect of the Lec35 mutation on glycosylphosphatidylinositol (GPI) lipid biosynthesis, another pathway that requires MPD. Our data indicate that the first mannosylation reaction of GPI lipid biosynthesis is defective in Lec35 cells, with the accumulation of glucosaminylphosphatidylinositol having a fatty acyl group on inositol and a base-resistant alkyl group attached to glycerol. The same intermediate accumulates in Lec15 (MPD synthase-defective) cells. The defective mannosylation reaction of Lec35 cells was corrected in vitro and shown to require MPD. These results demonstrate that the Lec35 gene governs a general aspect of MPD metabolism affecting both GPI lipid and dolichol-P-P-oligosaccharide biosynthesis. To provide additional insight into the role of the Lec35 gene, we give evidence for an inefficient pool of MPD in Lec35 membranes.