beta-1,6-Glucan plays a key structural role in the yeast cell wall. Of the genes involved in its biosynthesis, the activity of Cwh41p is known, i.e., the glucosidase I enzyme of protein N-chain glucose processing. We therefore examined the effects of N-chain glucosylation and processing mutants on beta-1,6-glucan biosynthesis and show that incomplete N-chain glucose processing results in a loss of beta-1,6-glucan, demonstrating a relationship between N-chain glucosylation/processing and beta-1,6-glucan biosynthesis. To explore the involvement of other N-chain-dependent events with beta-1,6-glucan synthesis, we investigated the Saccharomyces cerevisiae KRE5 and CNE1 genes, which encode homologs of the "quality control" components UDP-Glc:glycoprotein glucosyltransferase and calnexin, respectively. We show that the essential activity of Kre5p is separate from its possible role as a UDP-Glc:glycoprotein glucosyltransferase. We also observe a approximately 30% decrease in beta-1,6-glucan upon disruption of the CNE1 gene, a phenotype that is additive with other beta-1,6-glucan synthetic mutants. Analysis of the cell wall anchorage of the mannoprotein alpha-agglutinin suggests the existence of two beta-1,6-glucan biosynthetic pathways, one N-chain dependent, the other involving protein glycosylphosphatidylinositol modification.