Neisseria gonorrhoeae is a strict human pathogen that invades and colonizes the urogenital tracts of males and females. Lipooligosaccharide (LOS) has been shown to play a role in gonococcal pathogenesis. The acyl transferase MsbB is involved in the biosynthesis of the lipid A portion of the LOS. In order to determine the role of an intact lipid A structure on the pathogenesis of N. gonorrhoeae, the msbB gene was cloned and sequenced, a deletion and insertion mutation was introduced into N. gonorrhoeae, and the mutant strain was designated 1291A11K3. Mass spectrometric analyses of 1291A11K3 LOS determined that this mutation resulted in a pentaacyl rather than a hexaacyl lipid A structure. These analyses also demonstrated an increase in the phosphorylation of lipid A and an increase in length of the oligosaccharide of a minor species of the msbB LOS. The interactions of this mutant with male urethral epithelial cells (uec) were examined. Transmission and scanning electron microscopy studies indicated that the msbB mutants formed close associations with and were internalized by the uec at levels similar to those of the parent strain. Gentamicin survival assays performed with 1291A11K3 and 1291 bacteria demonstrated that there was no difference in the abilities of the two strains to adhere to uec; however, significantly fewer 1291A11K3 bacteria than parent strain bacteria were recovered from gentamicin-treated uec. These studies suggest that the lipid A modification in the N. gonorrhoeae msbB mutant may render it more susceptible to innate intracellular killing mechanisms when internalized by uec.