A locus involved in the biosynthesis of gonococcal lipooligosaccharide (LOS) has been cloned from gonococcal strain F62. The locus contains five open reading frames. The first and second reading frames are homologous, but not identical, to the fourth and fifth reading frames, respectively. Interposed is an additional reading frame which has distant homology to the Escherichia coli rfaI and rfaI genes, both glucosyl transferases involved in lipopolysaccharide core biosynthesis. The second and fifth reading frames show strong homology to the lex-1 or lic2A gene of Haemophilus influenzae, but do not contain the CAAT repeats found in this gene. Deletions of each of these five genes, of combinations of genes, and of the entire locus were constructed and introduced into parental gonococcal strain F62 by transformation. The LOS phenotypes were then analyzed by SDS-PAGE and reactivity with monoclonal antibodies. Analysis of the gonococcal mutants indicates that four of these genes are the glycosyl transferases that add GalNAc beta 1-->3Gal beta 1-->4GlcNAc beta 1-->3 Gal beta 1--4 to the substrate Glc beta 1-->4Hep--R of the inner core region. The gene with homology to E. coli rfaI/rfaI is involved with the addition of the alpha-linked galactose residue in the biosynthesis of the alternative LOS structure Gal alpha 1-->4Gal beta 1-->4Glc beta 1-->4Hep-->R. Since these genes encode LOS glycosyl transferases they have been named lgtA, lgtB, lgtC, lgtD, and lgtE. The DNA sequence analysis revealed that lgtA, lgtC, and lgtD contained poly-G tracts, which, in strain F62 were, respectively, 17, 10, and 11 bp. Thus, three of the LOS biosynthetic enzymes are potentially susceptible to premature termination by reading frame changes. It is likely that these structural features are responsible for the high-frequency genetic variation of gonococcal LOS.