Mouse monoclonal antibodies (MAbs) were derived against acetone-treated whole cells of the newly recognized Vibrio cholerae 0139 serogroup which is causing epidemics of cholera-like disease in India and Bangladesh. Four MAbs specifically recognized the lipopolysaccharide antigens of V. cholera 0139. Mabs ICL9 and ICL13 were of the immunoglobulin M (IgM) isotype, ICL11 was of the IgG3 isotype, and ICL12 was of the Ig2b isotype. A fifth MAb, ICL10, of the IgG2b isotype cross-reacted with V. cholerae 091. All five MAbs recognized V. cholerae 0139 in an enzyme-linked immunosorbent assay, slide agglutination test, motility inhibition test, and indirect immunofluorescence test. During a 1-month evaluation of these MAbs in our clinical laboratory, all 86 cases diagnosed as V. cholerae 0139 by a rabbit polyclonal antiserum were also detected by these MAbs, establishing their utility as highly sensitive and specific diagnostic reagents. With these MAbs, it should now be possible to screen for the V. cholerae 0139 serogroup in epidemic and endemic diarrhea cases and in environmental and food samples.