A highly penicillin-resistant beta-lactamase-producing strain of Clostridium butyricum, strain NBL 3, was isolated. The specific activity of the unpurified beta-lactamase was 0.29 U/mg of protein. The enzyme was purified 1,130-fold by QAE Zetaprep, Sephacryl S-300, and Mono Q column passages. The purified enzyme was judged homogeneous by sodium dodecyl sulfate gradient gel electrophoresis and fast protein liquid chromatography. The enzyme hydrolyzed phenoxymethylpenicillin, benzylpenicillin, ampicillin, and carbenicillin more rapidly than piperacillin and cephaloridine. Cephalothin, cephalexin, cefoxitin, moxalactam, cefotaxime, and imipenem were only slightly hydrolyzed, at less than 1% the rate for phenoxymethylpenicillin. The enzyme was inhibited by clavulanic acid, sulbactam, tazobactam, and p-chloromercuribenzoate. The molecular weight was determined by gel filtration and sodium dodecyl sulfate gradient gel electrophoresis to be 32,000. The isoelectric point was 4.4. Aspartic acid-asparagine, glutamic acid-glutamine, leucine, lysine, alanine, and serine dominated the amino acid composition.