An aminopeptidase isolated from the cytoplasmic fraction of a cell extract of Streptococcus mitis ATCC 903 was purified 330-fold by ion-exchange chromatography, gel filtration, and hydroxyapatite chromatography. The partially purified enzyme had a broad substrate specificity. Twelve aminoacyl-beta-naphthylamide substrates were hydrolyzed and also several di-, tri-, tetra-, and pentapeptides and bradykinin. The enzyme hydrolyzed arginine-beta-naphthylamide at the highest rate. Optimal conditions for activity were at pH 7.0-7.2 and at 37-40-degrees-C. The molecular weight of the enzyme was estimated to be 93,000. The enzyme was activated by Co2++ ions. Hg2+ inhibited the activity completely. SDS, EDTA, urea, and pCMB also inhibited activity. Inhibition by EDTA could be completely reversed by dialysis and addition of Co2+ ions. Reducing agents, sodium fluoride, and PMSF had no effect on the activity of the enzyme. The isoelectric point of the enzyme was at pH 4.3. High substrate concentrations inhibited activity. Substrate inhibition increased in the presence of high concentrations of Co2+ ions.