Monoclonal antibodies (MoAbs) to the lipopolysaccharide (LPS) of Bacteroides fragilis were produced by immunizing mice before hybridization with bacterial outer membranes solubilized with Triton X-100. Nineteen stabile clones were established. They all produced antibodies that reacted more strongly with purified B. fragilis LPS than with crude sonicated antigen in an enzyme immunoassay. Four MoAbs were studied by immunoblotting and enzyme immunoassay inhibition. Immunoblotting confirmed that the target of the MoAbs was LPS. Marked and homogeneous staining occurred in the immunoblotting with both purified LPS and outer membranes in the molecular weight range of 8,000 to 27,000. In enzyme immunoassay inhibition, MoAbs reacted positively with 93 to 96% of B. fragilis strains, including prototype strains ATCC 23745 and NCTC 9343. Within the B. fragilis group, the MoAbs reacted positively with two of five B. ovatus strains and two to six of nine B. thetaiotaomicron strains. No marked cross-reactivity with other bacteria was observed. These results confirm earlier findings that the B. fragilis LPS contains an immunodominant antigenic determinant common to almost all B. fragilis isolates.