The β-lactamases excreted by the highly benzylpenicillin-susceptible Streptomyces strain R39 and the highly benzylpenicillin-resistant Streptomyces albus G were isolated and purified. Neither β-lactamase exhibited dd -carboxypeptidase activity. Both were anionic at p H 8.3, did not require metal ions, and were not sensitive to iodine, but were inhibited by Cu 2+ and readily inactivated by heat. p -Chloromercuribenzoate, iodoacetate, p -aminobenzoate, and substrates and inhibitors of dd -carboxypeptidase had no effect on β-lactamase activity. The K m and V max values for β-lactamase activity were studied with 6-aminopenicillanic acid and with various penicillins and cephalosporins. The β-lactamase from the related strain K11 of Streptomyces , which is intermediate in its susceptibility to benzylpenicillin, was partially purified, and its activity was compared on the various substrates.