Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of purified lipopolysaccharide (LPS) from Neisseria gonorrhoeae resulted in the formation of multiple bands. Many of the bands consisted of LPS aggregates, which could be dissociated by treatment with 0.1 M NaOH or by addition of 4 M urea to the separating gel. The unaggregated LPS was usually found in one to three bands toward the bottom of the gels, a result suggesting that a long repeating O antigen is not present on gonococcal LPS. SDS-PAGE of LPS from different LPS serotypes of N gonorrhoeae indicated that structural heterogeneity exists. Antigenic analysis by enzyme-linked immunosorbent assay inhibition of gonococcal LPS extracted with phenol-chloroform-petroleum ether (PCP) and phenol-water revealed that PCP-extracted LPS contained substantially less serotype-specific antigen than did phenol-water-extracted LPS. These results suggest that the PCP and phenol-water methods extract different molecular species of LPS from N gonorrhoeae.