Penicillin-binding proteins (PBPs) of Treponema pallidum subsp. pallidum (T. pallidum) were characterized by using [3H]penicillin G and a conjugate consisting of ampicillin and 125I-labeled Bolton-Hunter reagent. Both antibiotics specifically radiolabeled proteins with molecular masses of 94, 80, 63, and 58 kilodaltons (kDa); 125I-labeled Bolton-Hunter reagent-ampicillin also radiolabeled several polypeptides with lower molecular masses. The 94- and 58-kDa proteins demonstrated the highest binding affinities for [3H]penicillin G and were radiolabeled at concentrations of 8 and 40 nM, respectively. Radiolabeling of PBPs was detectable after 1 min of incubation in 1 microM [3H]penicillin G and was nearly maximal within 10 min. The rapidity of penicillin binding contrasted with the observation that only 40% of virulent treponemes became immobilized during prolonged incubation in vitro with a much higher concentration (1 mM) of unlabeled penicillin. Two lines of evidence indicated that most, if not all, of the PBPs are integral cytoplasmic membrane proteins: (i) preincubation of organisms in 0.1% Triton X-100 solubilized nearly all of the outer membranes but did not affect radiolabeling of PBPs, and (ii) except for the 80-kDa protein, the PBPs partitioned into the detergent phase following extraction with the nonionic detergent Triton X-114. The presence of peptidoglycan in T. pallidum was confirmed by the detection of muramic acid in the sodium dodecyl sulfate-insoluble, proteinase K-resistant residue obtained from Triton X-114-extracted organisms.