CWH41, a gene involved in the assembly of cell wall beta-1,6-glucan, has recently been shown to be the structural gene for Saccharomyces cerevisiae glucosidase I that is responsible for initiating the trimming of terminal alpha-1,2-glucose residue in the N-glycan processing pathway. To distinguish between a direct or indirect role of Cwh41p in the biosynthesis of beta-1,6-glucan, we constructed a double mutant, alg5Delta (lacking dolichol-P-glucose synthase) cwh41Delta, and found that it has the same phenotype as the alg5Delta single mutant. It contains wild-type levels of cell wall beta-1,6-glucan, shows moderate underglycosylation of N-linked glycoproteins, and grows at concentrations of Calcofluor White (which interferes with cell wall assembly) that are lethal to cwh41Delta single mutant. The strong genetic interactions of CWH41 with KRE6 and KRE1, two other genes involved in the beta-1,6-glucan biosynthetic pathway, disappear in the absence of dolichol-P-glucose synthase (alg5Delta). The triple mutant alg5Deltacwh41Deltakre6Delta is viable, whereas the double mutant cwh41Deltakre6Delta in the same genetic background is not. The severe slow growth phenotype and 75% reduction in cell wall beta-1,6-glucan, characteristic of the cwh41Deltakre1Delta double mutant, are not observed in the triple mutant alg5Deltacwh41Deltakre1Delta. Kre6p, a putative Golgi glucan synthase, is unstable in cwh41Delta strains, and its overexpression renders these cells Calcofluor White resistant. These results demonstrate that the role of glucosidase I (Cwh41p) in the biosynthesis of cell wall beta-1,6-glucan is indirect and that dolichol-P-glucose is not an intermediate in this pathway.