An inverse relationship between binding of [(14)C]penicillin to cells of Neisseria gonorrhoeae and the minimal inhibitory concentration of penicillin was recently reported by us. Herein we report a similar relationship in binding by cytoplasmic membranes derived from 10 strains of gonococci. Membranes were extracted after Braun homogenization and differential centrifugation of the resulting extracts. Membranes derived from penicillin-susceptible cells bound 140 to 200 nmol of [(14)C]penicillin per g of enzyme, whereas relatively nonsusceptible strains bound 18 to 59 nmol. Binding by cell walls was only a small fraction of the total binding and probably represents contamination of the walls by membrane. Pretreatment of cell membranes with either unlabeled penicillin or cephalothin prior to [(14)C]penicillin exposure markedly decreased (14)C binding. Slab gel electrophoresis of membranes revealed 13 protein bands, 8 of which were labeled.