Penicillin-binding proteins (PBPs) of Listeria monocytogenes were detected by their ability to bind to [2,3-3H]benzylpenicillin. Five proteins with M,s of 95,000, 84,000, 80,000, 76,000, and 49,000 were detected. PBPs 1 to 4 had a high affinity for [2,3-3H]benzylpenicillin and were relatively scarce (80 to 150 molecules per cell). In contrast, PBP 5 was more abundant (600 molecules per cell) but had a low affinity for [2,3-3Hlbenzylpenicillin. L. monocytogenes has a relatively high natural resistance to cephalosporins. Competition experiments showed that cephalosporins bound very poorly to PBP 3 but were good inhibitors of PBPs 1, 2, and 4, which were completely blocked at concentrations well below the MIC. Analysis of a spontaneous imipenem-resistant mutant revealed that resistance was likely due to an altered PBP 3 with a reduced affinity for [2,3-3H]benzylpenicillin. These results suggest that PBP 3 is a primary lethal target for ,-lactams in L.monocytogenes.