An aminopeptidase from Streptococcus sanguis NCTC 10904 was isolated, purified, and characterized. The enzyme was produced constitutively and could be isolated from the cytoplasmic fraction of lysed cells. Its substrate profile indicated that it is primarily a leucyl aminopeptidase, but with a substrate spectrum including lysyl- and arginyl-peptides. The subunit molecular weight of the enzyme was approximately 74,000, but an octomeric form also was prominent, as indicated by gel filtration separations of active enzymes. The optimal temperature for activity was 32-degrees-C, and the optimal pH value was about 7.0. The enzyme showed cooperative kinetics and was activated by Co2+. The regulation of synthesis and the characteristics of the enzyme suggest that it may serve a regulatory function rather than just a nutritional function.