A soluble α-mannosidase from Candida albicans was purified to homogeneity by sequential size exclusion, ion exchange, and affinity chromatographies in columns of Sepharose CL6B, DEAE Bio-Gel A, and Concanavalin A Sepharose 4B, respectively. Analytical electrophoresis of the purified preparation in 10% SDS–polyacrylamide gels stained with Coomassie blue revealed a single polypeptide of 43 kDa that was responsible for enzyme activity. The purified enzyme primarily trimmed Man9GlcNAc2 to produce Man8GlcNAc2 isomer B and mannose as a function of time of incubation up to 12 h at 37°C. Prolonged incubation with the enzyme resulted in the accumulation after 24 h of other oligosaccharides corresponding to Man7GlcNAc2 and probably Man6GlcNAc2. These two products were also observed when Man8GlcNAc2 isomer B instead of Man9GlcNAc2 was used as substrate. Other oligosaccharides, such as Man6GlcNAc2-Asn, Man5GlcNAc2-Asn, and the α1,3- and α1,6-linked mannobiosides, were not hydrolyzed at all. These properties are consistent with an α1,2-mannosidase that may represent a new member of the glycosylhydrolase family 47.