An enzyme-linked immunosorbent assay (ELISA) was developed to detect specific antibodies against Mycobacterium paratuberculosis in bovine serum. Experiments were designed to find methods to avoid the false-positive reactions that frequently are encountered in ELISA for the detection of antibodies to protoplasmic antigen of M.paratuberculosis. Sera examined were obtained from cattle infected with M.paratuberculosis, Mycobacterium bovis, Mycobacterium kansasii, or Nocardia asteroides, from cattle which were negative in bacteriological examination but positive in the complement-fixation test for paratuberculosis, and from sheep infected with Corynebacterium pseudotuberculosis. Preabsorption of tested sera with Mycobacterium phlei resulted in substantially eliminating false-positive reactions. This absorption treatment had no effect on the ELISA antibody level of sera from cattle infected with M.paratuberculosis. These results indicate that the false-positive reactions that occur in the ELISA test for the diagnosis of bovine paratuberculosis can be controlled by preabsorption of the tested serum with M phlei.