Protective immunity against shigellosis is thought to be determined by the O-antigen side chains of the lipopolysaccharide (LPS) molecule. To study possible common protective epitopes, monoclonal antibodies reacting with Shigella flexneri 2a LPS were generated from BALB/c mice infected ocularly with the virulent serotype 2a strain S. flexneri 2457T and tested against a panel of S. flexneri LPSs by enzyme-linked immunosorbent and immunoblot assays. Four monoclonal antibodies were identified, all of which showed restricted specificity patterns. Three different patterns of reactivity to LPS possessing the 3,4 group antigen were seen: (i) 2a only, (ii) 2a and 5a, and (iii) 2a, 4a, 5a, and Y. These results have implications for designing a Shigella vaccine that will be protective against related serotypes. Electron microscopy studies showed that the monoclonal antibodies bind to the bacterial surface in a patchy pattern, suggesting their potential use for examining the LPS distribution on the surface of the bacteria.