Lec15 and Lec35 are recessive Chinese hamster ovary (CHO) cell glycosylation mutations characterized by inefficient synthesis and utilization, respectively, of mannose-P-dolichol (MPD). Consequently, Lec15 and Lec35 cells accumulate Man5GlcNAc2-P-P-dolichol and glucosaminyl-acylphosphatidylinositol. This report describes the cloning of a suppressor (termed SL15) of the Lec15 and Lec35 mutations from a CHO cDNA library by functional expression in Lec15 cells, employing phytohemagglutinin/swainsonine selection. The SL15 protein has a predicted molecular weight of 26,693 with two potential membrane spanning regions and a likely C-terminal endoplasmic reticulum retention signal (Lys-Lys-Glu-Gln). Lec15 cells transfected with SL15 have normal levels of MPD synthase activity in vitro and convert Man5GlcNAc2-P-P-dolichol to Glc0-3Man9GlcNAc2-P-P-dolichol in vivo. Surprisingly, SL15 also corrects the defective mannosylation in Lec35 cells. The SL15 protein bears no apparent similarity to Saccharomyces cerevisiae MPD synthase (the DPM1 protein), but is highly similar to the hypothetical F38E1.9 protein encoded on Caenorhabditis elegans chromosome 5. These results indicate a novel function for the SL15 protein and suggest that MPD synthesis is more complex than previously suspected.