A collection of wild-type and reference strains of Streptococcus mutans and Streptococcus sobrinus was compared using a set of fermentation and enzymic tests in order to establish a simple scheme for the differentiation of these two species isolated from selective media. Tests for the production of acid from N-acetylglucosamine, arbutin and melibiose, and the presence of alpha-galactosidase and alpha-glucosidase activities were useful in differentiating these species. Strains of Streptococcus mutans unable to ferment melibiose were also frequently unable to ferment other sugars; a consideration of the genetic control and organization of the genes involved in melibiose metabolism indicates that certain of the conventional characterization tests are not measurements of independent biochemical capabilities.