The precise ultrastructural localization of penicillin-binding protein (PBP)-antibiotic complexes in Escherichia coli JM101, JM101 (pBS96), and JM101(pPH116) was investigated by high-resolution electron microscopy. We used mercury-penicillin V (Hg-pen V) as a heavy-metal-labeled, electron-dense probe for accurately localizing PBPs in situ in single bacterial cells grown to exponential growth phase. Biochemical data derived from susceptibility tests and bacteriolysis experiments revealed no significant differences between Hg-pen V and the parent compound, penicillin V, or between strains. Both antibiotics revealed differences in the binding affinities for PBPs of all strains. Deacylation rates for PBPs were slow despite the relatively low binding affinities of antibiotics. Cells bound most of the Hg-pen V added to cultures, and the antibiotic-PBP complex could readily be seen by electron microscopy of unstained whole mounts as distinct, randomly situated electron-dense particles. Fifty to 60% of the antibiotic was retained by cells during processing for conventional embedding so that thin sections could also be examined. These revealed similar electron-dense particles located predominantly on the plasma membrane and less frequently in the cytoplasm. Particles positioned on the plasma membranes were occasionally shown to protrude into the periplasmic space, thereby reflecting the high resolution of the Hg-pen V probe. Moreover, some particles were observed free in the periplasm, suggesting, for the first time, that a proportion of PBPs may not be restricted to the plasma membrane but may be tightly associated with the peptidoglycan for higher efficiency of peptidoglycan assembly. All controls were devoid of the electron-dense particles. The presence of electron-dense particles in cells of the wild-type JM101, demonstrated that our probe could identify PBPs in naturally occurring strains without inducing PBP overproduction.