An arginine carboxypeptidase was isolated from the cell walls of Streptococcus mitis ATCC 15909 by mutanolysin extraction of the walls. The enzyme was purified 32-fold by gel filtration on Sephacryl S-300, affinity chromatography on Arginine-Sepharose 4B and by rechromatography on Sephacryl S-300. The molecular mass of the enzyme was calculated to 122 kDa by gel filtration. The enzyme released arginine from the carboxy terminal of hippuryl-arginine and, at a low rate, lysine from furylacrylolylalanyl-lysine and hippuryl-lysine. The carboxypeptidase seemed firmly bound to the cell wall because SDS treatment of the walls did not release measurable amounts of activity.