Twenty-three penicillinase-producing strains of Neisseria gonorrhoeae were characterized. All strains showed hybridization with a TEM-1 probe and production of a TEM-1 type beta-lactamase (pI 5.4) on isoelectric focusing. Eight strains also showed a beta-lactamase band of pI 5.5. The penicillin MICs for the strains producing only TEM-1 were in the range 4-16 micrograms/ml; MICs of the strains with the additional pI 5.5 band were > or = 128 micrograms/ml for seven strains and 4 micrograms/ml for an arginine-hypoxanthine dependent strain. The association of the high MICs with the presence of the pI 5.5 band was statistically significant. The pI 5.5 band could represent a new beta-lactamase type or a TEM-1 mutant.