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Whole-cell lysate and proteinase K digest preparations of the Mycoplasma bovis type strain (American Type Culture Collection 25523) were compared using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Coomassie blue staining for protein revealed approximately 50 bands for the lysate but only a single band for the digest. Silver staining for polysaccharide revealed at least 19 bands for the digest. Fourteen monoclonal antibodies (MAbs) were produced using a screening procedure with an M. bovis digest. On immunoblots of digests of four M. bovis strains, an almost identical profile was seen with each strain for all 14 MAbs but differences were evident between strains. One MAb, M1557, was used to analyse 17 M. bovis strains on immunoblots. Ten to 20 bands were observed with 16 of the 17 strains, and differences were apparent among all 16 strains. In an enzyme-linked immunosorbent assay, M1557 reacted with 16 of the 17 M. bovis strains, but did not react with any of 41 non-M. bovis organisms tested. Strong reactions were observed with the MAbs and M. bovis colonies in immunofluorescence. The M. bovis polysaccharide and MAbs to this component may be useful for the development of diagnostic assays for this organism.