The non-enteric Gram-negative human pathogens, B. catarrhalis, H. ducreyi, H. influenzae, N. gonorrhoeae and N. meningitidis, do not have repeating O-antigens as part of their principle surface glycolipid, the lipooligosaccharide (LOS). Because they have similar LOS structures, we studied the conservation of LOS oligosaccharide epitopes among these organisms. Twenty-one monoclonal antibodies (mAbs) generated by immunizing mice with H. influenzae, N. gonorrhoeae and N. meningitidis were studied for cross reactivity. Five mAbs generated against non-typable H. influenzae were the only strain-specific antibodies. Ten mAbs reacted to LOS epitope(s) common to a genera or species, and six mAbs bound to epitope(s) on the LOS of strains from different genera. Some cross reactive mAbs bound to LOS bands of similar molecular weights, while others bound to bands of varying molecular weights. mAb 3F11, whose epitope mimics a human blood-group antigen, bound to a 4.8 kDa LOS band in N. gonorrhoeae and H. ducreyi, two pathogens that infect genital epithelium. mAb 3D9, whose epitope consists of 2-keto-3-deoxyoctulosonic acid (KDO), reacted with different LOS bands in N. gonorrhoeae, H. influenzae and some R mutants of S. minnesota. A 14 kb restriction fragment containing lipooligosaccharide synthesis genes responsible for the assembly of the 3D9 epitope in H. influenzae hybridized to all H. influenzae strains tested but did not hybridize to gonococcal and S. minnesota strains that expressed this epitope. These studies demonstrate that conserved LOS epitope(s) exist among different species and genera of non-enteric human pathogens and that different genetic mechanisms may have evolved in these pathogens to assemble some of these conserved epitopes.