The specificity of an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of paratuberculous cattle was improved by absorbing the bovine sera with a suspension of Mycobacterium phlei-killed organisms. The newly developed ELISA technique was evaluated and compared with the complement fixation (CF) test for the detection of antibodies to Mycobacterium paratuberculosis. Thirty-eight (66%) of 58 fecal culture positive cattle in a M. paratuberculosis infected herd were positive in the ELISA test, whereas the CF test yielded only 15 (26%) positive results. Calves experimentally infected with M. paratuberculosis seroconverted in the ELISA test at the earlier stage of infection than in the CF test. The ELISA was proposed as an alternative to the CF test for the detection of antibodies against M. paratuberculosis in bovine serum. Application of the ELISA as a diagnostic test for subclinical infection of cattle with M. paratuberculosis was proved to be very useful.