1. Mycobacterium smegmatis (N.C.T.C. 8158), M. fortuitum and M. phlei (MPI) produce a constitutive β-lactamase that has penicillinase and cephalosporinase activity. 2. The β-lactamases of these three species of acid-fast bacteria were mainly cell-bound, only small amounts of activity being liberated into the extracellular fluid. The total β-lactamase activity of these mycobacteria was much lower than that of certain Gram-positive organisms, but comparable with that reported for species of Gram-negative bacteria. 3. The β-lactamases of intact cells of the mycobacteria were not freely accessible to any of the substrates tested, but the apparent crypticity factor to benzylpenicillin was greater than that to cephaloridine and cephalosporin C. 4. Attempts to induce β-lactamase activity in M. smegmatis and M. phlei failed even with high concentrations of inducer. 5. The β-lactamases obtained from the three species of mycobacteria showed different substrate specificities, including different relative activities as cephalosporinases and penicillinases respectively. 6. Certain derivatives of 6-aminopenicillanic acid and 7-aminocephalosporanic acid were found to be resistant to hydrolysis by β-lactamases of M. smegmatis and M. fortuitum. 7. The β-lactamase of M. smegmatis was competitively inhibited by a number of β-lactamase-resistant derivatives of 6-aminopenicillanic acid, but not by similar derivatives of 7-aminocephalosporanic acid.
