Mycobacteria produce beta-lactamases and are intrinsically resistant to beta-lactam antibiotics. In addition to the beta-lactamases, cell envelope permeability and variations in certain peptidoglycan biosynthetic enzymes are believed to contribute to beta-lactam resistance in these organisms. To allow the study of these additional mechanisms, mutants of the major beta-lactamases, BlaC and BlaS, were generated in the pathogenic Mycobacterium tuberculosis strain H37Rv and the model organism Mycobacterium smegmatis strain PM274. The mutants M. tuberculosis PM638 (DeltablaC1) and M. smegmatis PM759 (DeltablaS1) showed an increase in susceptibility to beta-lactam antibiotics, as determined by disc diffusion and minimal inhibitory concentration (MIC) assays. The susceptibility of the mutants, as assayed by disc diffusion tests, to penicillin-type beta-lactam antibiotics was affected most, compared to the cephalosporin-type beta-lactam antibiotics. The M. tuberculosis mutant had no detectable beta-lactamase activity, while the M. smegmatis mutant had a residual type 1 beta-lactamase activity. We identified a gene, blaE, encoding a putative cephalosporinase in M. smegmatis. A double beta-lactamase mutant of M. smegmatis, PM976 (DeltablaS1DeltablaE : : res), had no detectable beta-lactamase activity, but its susceptibility to beta-lactam antibiotics was not significantly different from that of the DeltablaS1 parental strain, PM759. The mutants generated in this study will help determine the contribution of other beta-lactam resistance mechanisms in addition to serving as tools to study the biology of peptidoglycan biosynthesis in these organisms.