β-Lactamase fromMycohacterium smegamatis SN2 was purified to homogeneity. The molecular weight of the enzyme was 30,000 and the isoelectric point was 4.1. The enzyme showed maximal activity at pH 6.5 and 56°C and resembled the plasmid-mediated TEM-type β-lactamases commonly encountered in gram-negative bacteria in substrate profile. The enzyme shared antigenic structure with β-lactamase fromMycobacterium butyricum ATCC 19979 andEscherichia coli HB101 (pBR322).