The genetic relatedness of 354 non-penicillinase-producing Neisseria gonorrhoeae strains was studied by multilocus enzyme electrophoresis. The strains, isolated over 9 years in Spain, had different levels of susceptibility to penicillin and tetracycline. On the basis of results obtained by analysis at 12 enzyme loci, the strains were separated into 46 electrophoretic types (ETs). The average number of alleles per enzyme locus was 2.07, and the mean genetic diversity per locus was 0.26 for ETs and 0.27 for individual isolates.The distribution of strains among ETs was not linked to any particular auxotype or serovar class. The index of discrimination of auxotyping combined with serovar determination was similar to multilocus enzyme analysis. Classification of strains by auxotype and serovar did not seem to be related to genotype. Clustering analysis revealed that moderate penicillin-resistant and chromosomally mediated tetracycline-resistant strains were largely shared, while penicillin-resistant strains appeared only in some clusters.