The naphthylamidase isozyme complement ofSarcina luteawas studied. Gel filtration yielded two fractions, Sephadex I and Sephadex II. Sephadex I contained one enzyme generally resembling leucineaminopeptidase. Sephadex II, upon ion exchange chromatography, yielded three isozymes, A, B, and C. These three were characterized with respect to molecular weight, substrate specificities, and effects of hydrogen ion concentration, EDTA, and divalent cation on reaction velocity. The molecular weights are 8.0 × 104, 8.2 × 104, and 9.0 × 104respectively. Isozymes A and B are neutral naphthylamidases and preferentially catalyze the hydrolysis of alanine-β-naphthylamide (βNA), whereas isozyme C is a basic naphthylamidase and preferentially catalyzes the hydrolysis of lysine and arginine-βNA. The pH optima for the isozymes are 7.6, 7.6, and 6.7, respectively. All of the isozymes are sensitive to the effects of EDTA. Divalent cations activate the enzymes and reverse inhibition caused by EDTA.