Summary: The aga gene coding for α-galactosidase in Streptococcus mutans was detected in a recombinant gene library constructed in phage λ. The gene was subcloned into plasmid vectors and shown to specify a novel protein of Mr 80000. Characterization of α-galactosidase from S. mutans and from recombinant Escherichia coli expressing aga indicated that the enzyme functions as a tetramer. The amino acid composition of the α-galactosidase, deduced from nucleotide sequencing of aga, gave a predicted Mr of 82022 and revealed regions of homology to α-galactosidases encoded by the E. coli Raf plasmids and by Bacillus stearothermophilus. Inactivation of the aga gene in S. mutans resulted in loss of all α-galactosidase activity and abolished the ability to ferment melibiose; α-glucosidase activity was also lost, due to an indirect effect on the dexB gene.