The membrane (M) protein of canine coronavirus (CCoV) was cloned and expressed in E. coli. The purified recombinant protein was then evaluated for its antigenicity and reliability in an enzyme-linked immunosorbent assay (ELISA) for detection of CCoV antibodies in dog sera. Fifty serum samples, screened previously by whole virus ELISA and Western blotting, were tested. When the performance of the new test was compared with those of whole virus ELISA and Western blotting, an excellent correlation was found with the latter two assays. The ELISA based on recombinant M protein represents an alternative and valid test for detection of antibodies to CCoV in dog sera.