A HindIII gene bank of Neisseria gonorrhoeae MUG116 was constructed in the cosmid vector pHC79. A cosmid (pSY81) was isolated that was able to convert N. gonorrhoeae FA5100 to reactivity with monoclonal antibody (MAb) 2-1-L8. Several MAb-reactive transformants were isolated and characterized with respect to lipooligosaccharide (LOS) production as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, their ability to react with two other LOS-specific MAbs (3F11 and O6B4), and Southern blot analysis. Escherichia coli containing the clone had altered lipopolysaccharide expression as determined by electrophoretic analysis; however, no reactivity was seen with gonococcus-specific MAbs. The introduction of pSY81 into FA5100 had a pleiomorphic effect, giving rise to transformants having the full parental phenotype or transformants lacking reactivity to a combination of LOS-specific MAbs. Southern blot analysis indicated that the LOS biosynthetic mutation in FA5100 was not due to chromosomal rearrangement or large deletions.