Previously a Brucella protein named CP28, BP26, or Omp28 has been identified as an immunodominant antigen in infected cattle, sheep, goats, and humans. In the present study we evaluated antibody responses of infected and B. melitensis Rev.1-vaccinated sheep to the BP26 protein using purified recombinant BP26 protein produced in Escherichia coli in an indirect enzyme-linked immunosorbent assay (I-ELISA). The specificity of the I-ELISA determined with sera from healthy sheep (n = 106) was 93%. The sensitivity of the I-ELISA assessed with sera from naturally infected and suspected sheep found positive in the current conventional diagnostic tests was as follows: 100% for bacteriologically and serologically positive sheep (n = 50), 88% for bacteriologically negative but serologically and delayed-type hypersensitivity-positive sheep (n = 50), and 84% for bacteriologically and serologically negative but delayed-type hypersensitivity-positive sheep (n = 19). However, the absorbance values observed did not reach those observed in an I-ELISA using purified O-polysaccharide (O-PS) as an antigen. In sheep experimentally infected with B. melitensis H38 the antibody response to BP26 was delayed and much weaker than that to O-PS. Nevertheless, the BP26 protein appears to be a good diagnostic antigen to be used in confirmatory tests and for serological differentiation between infected and B. melitensis Rev.1-vaccinated sheep. Weak antibody responses to BP26 in some of the latter sheep suggest that a B. melitensis Rev.1 bp26 gene deletion mutant should be constructed to ensure this differentiation.