The minimal inhibitory concentrations (MICs) of ampicillin for fifty strains of beta-lactamase-producing Neisseria gonorrhoeae (PPNG) isolated in Japan ranged from 1.56 to 200 micrograms/ml, and all the strains harbored a 4.5 megadalton plasmid. These strains were classified into two groups: dicloxacillin-susceptible (28%) and -resistant group (72%). A linear correlation was found in the dicloxacillin-susceptible strains between their beta-lactamase activity and the susceptibility to ampicillin, but not in the dicloxacillin-resistant strains. This suggests that the high ampicillin resistance in PPNG is due not only to acquiring the beta-lactamase producing plasmid, but also to some intrinsic resistance of the strains. To investigate a cause of the high ampicillin resistance, the beta-lactamase-producing plasmid, pTMS1, was transferred by conjugation to a penicillin-susceptible gonococcal strain as well as to its isogenic multiply antibiotic-resistant transformants, and the susceptibility of the transconjugants to ampicillin was determined. Acquisition of pTMS1 by a penicillin-susceptible strain resulted in a 32-fold increase in resistance to ampicillin, whereas the increase was 128-fold for its isogenic strains which contain some chromosomal mutations. These results suggest that reduced permeability of the outer membrane to ampicillin underlies the high ampicillin resistance of PPNG.