Genomic clones were isolated that code for three glycosidases proposed to be involved in the catabolism of cell wall components in Saccharomyces cerevisiae. alpha-Mannosidase (AMS1), exoglucanase (BGL1), and endochitinase (CTS1) genes were isolated with the aid of filter assays based on the hydrolysis of 4-methylumbelliferyl glycosides, which permitted the in situ monitoring of these glycosidase activities in yeast colonies. Uracil prototrophs resulting from transformation with a multicopy YEp24 yeast genomic library were screened, leading to the identification of transformants possessing high levels of glycosidase activity. Restriction maps of plasmids from multiple isolates were used to localize glycosidase-overproduction genes, which were subcloned into a Schizosaccharomyces pombe/S. cerevisiae shuttle vector. Transformation of Sch. pombe with BGL1 and CTS1 subclones resulted in the appearance of these activities in this organism, and an AMS1 plasmid caused a 2-fold increase in endogenous alpha-mannosidase levels. Insertion of the marker gene LEU2 into putative AMS1 sequences disrupted plasmid-encoded alpha-mannosidase overproduction. S. cerevisiae strains that incorporated a restriction fragment containing ams1::LEU2 into their chromosomal DNA by homologous recombination expressed no detectable alpha-mannosidase activity in either the haploid or homozygous recessive diploid states, whereas heterozygous and wild-type cells exhibited levels proportional to AMS1 gene dosage. No readily apparent phenotype was associated with the alpha-mannosidase deficiency; however, labeling experiments utilizing [2-3H]mannose suggest that alpha-mannosidase may function in mannan turnover.