By use of (14)C-labeled benzyl penicillin, it has been established that beta-lactamases and/or acylases play no role in the resistance of Neisseria gonorrhoeae to penicillin. It has been found, however, that very susceptible strains of the organisms (minimal inhibitory concentration, 0.008 mug/ml) bind 10 to 15 times as much penicillin as do moderately to highly resistant strains of the gonoccoccus (minimal inhibitory concentration, 0.125 to 2.0 mug/ml). It is postulated that this degree of change in binding components of the whole cell and whole cytoplasmic membrane is sufficient to account for the decreased susceptibility of the organism to penicillin.