A penicillin-resistant (Penr) non-penicillinase-producing Neisseria gonorrhoeae strain responsible for an outbreak affecting 199 persons in Durham, N.C., in 1983 was studied to determine the genetic basis of its unusually high-level (MIC, 2.0 micrograms/ml) Penr. Plasmid screening of the strain revealed no plasmids other than the 2.6-megadalton cryptic plasmid. Penr was found to be partially due to mutations genotypically and phenotypically similar to the previously characterized chromosomal loci penA, mtr, and penB. Resistance loci from the epidemic donor strain were transformed into susceptible recipients FA19 and F62 in a stepwise fashion; the combination of the three loci resulted in moderate levels of penicillin resistance (MIC, 0.5 micrograms/ml), but donor levels of resistance were not obtainable in either recipient, for uncertain reasons. Occurrence of an antibiotic-susceptible (env) mutation in a clinical isolate of the Penr epidemic strain also was documented.