Six hybridoma cell lines, each of which produced a monoclonal antibody (MAb) against Vibrio cholerae O1 lipopolysaccharide (LPS), were established. Each MAb was active serologically by both enzyme-linked immunosorbent assay (ELISA) and the slide agglutination test. In the ELISA, each MAb was tested against 7 O1 and 9 non-O1 LPS preparations. Three MAbs reacted with both Inaba and Ogawa serovars (A antigen), two MAbs reacted with the Ogawa serovars only (B antigen), and one MAb reacted with the Inaba serovars only (C antigen). Each MAb was also tested in the ELISA against whole-cell preparations of 37 O1 and 52 non-O1 V. cholerae serovars, 20 heterologous Vibrio species, and 37 heterologous bacterial species. The MAbs reacted with V. cholerae O1 cells only, except for one anti-A antigen MAb which reacted weakly with five V. cholerae non-O1 serovars and Serratia marcescens. Each anti-A antigen MAb was labeled with fluorescein isothiocyanate (FITC) and tested by direct immunofluorescence against selected O1 and non-O1 serovars. Each MAb-FITC conjugate, when tested alone, exhibited O1-specific fluorescence; however, mixtures of the MAb-FITC dramatically enhanced fluorescence intensity on O1 cells. This finding was also visualized by immunoelectron microscopy on both thin-sectioned and negatively stained O1 cells by using an anti-mouse immunoglobulin-colloidal gold conjugate. These results suggest that the A antigen can be described by more than one epitope and that a superior serotyping reagent can be prepared from a defined mixture of MAbs.