Sporobolomyces singularis can be used as a biocatalyst in galacto-oligosaccharide production. We isolated 2-deoxy-D-glucose-resistant mutants of S. singularis ATCC 24193 and recovered a mutant that showed 10-fold higher beta-galactosidase-like activity than the parent strain and which was insensitive to catabolite repression. Thereafter, the beta-galactosidase-like enzyme was purified from the mutant and revealed to be a glycoprotein with both beta-glucosidase- and beta-galactosidase-like activity, the Michaelis-Menten constants of which for o-nitrophenyl-beta-D-galactopyranoside and p-nitrophenyl-beta-D-glucopyranoside were 5.40 and 1.96 mM, respectively, and the maximum velocities were 3.07 and 2.30 micromol/min per mg of protein, respectively. Its molecular mass was estimated to be 73.9 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 146 kDa by gel filtration, suggesting that it has a homodimeric structure. We sequenced the N-terminus and internal peptides of this protein and isolated both a cDNA and a gene with degenerate primers. The gene, named bg l A, has 18 introns and 19 exons and encodes a polypeptide of 594 amino acids. The Bg l A protein was approximately 35% identical and 50% similar to plant beta-glucosidases belonging to family 1 glycosyl hydrolases, but with a unique 110-amino-acid sequence at the N-terminus. The beta-galactosidase-like enzyme (i.e., Bg l A protein) in S. singularis is a beta-glucosidase with high transgalactosidase activity.