This study describes the response of cattle to an enzyme-linked immunosorbent assay (ELISA) for paratuberculosis, using serum from each animal. The results of fecal culture confirmed clinical suspect cases were analyzed in relation to the amount of colonies isolated from the animals on fecal culture (0,+,++,+++,++++ and above). A significant increase in ELISA response in animals with heavy M. paratuberculosis shedding (++++ or above) using both unabsorbed and absorbed serum was found, as compared to animals that were fecal culture negative or shedding M. paratuberculosis at lower levels (++++), (P less than 0.05). The effects on sensitivity and specificity of using different cut-off points in the different test groups was described, since sera were not discretely segregated into distinct groups of positives and negatives. The specificity of the ELISA in two fecal culture negative herds was 100% at an ELISA cut-off of 0.1 optical density (O.D.) and above for absorbed serum. For unabsorbed serum the specificity was 62.9% of a similar cut-off value. Similarly the specificity of a fecal culture negative, serologic positive herd increased from 37.5 to 72.2 at an ELISA cut-off value of 0.1-0.2 (O.D.) using absorbed versus unabsorbed serum, and from 75.0 to 94.4 at an ELISA cut-off value of 0.2-0.3.