In order to associate virulence in Neisseria gonorrhoeae with an alteration of the nucleotide sequence of its small covalently closed plasmid, plasmid deoxyribonucleic acid was isolated from both virulent (T1) and avirulent (T3) morphological types for two strains. Electrophoretic and contour length measurements of intact plasmids indicated a homogeneous population with a molecular weight of approximately 2.6 x 10(6). Digestion with two restriction endonucleases. Hinf I and Hpa II, generated distinct fragment patterns which in each case were identical for T1 and T3 plasmid molecules from the same strain. The analysis suggests no sequence differences between the plasmids from virulent and avirulent types. For both strains, however, a deletion or addition of about 1.5% of the total deoxyribonucleic acid appeared in the Hpa II C digestion fragment when patterns for gonococci serially passaged 300 times were compared to those for bacteria freshly established from frozen stocks. The significance of the plasmid instability remains undetermined.