Two thousand nine hundred fifty-two serum samples, collected once or twice annually from 545 cows of known fecal culture status were tested for antibodies to Mycobacterium avium subsp. paratuberculosis using a commercially available enzyme-linked immunosorbent assay (ELISA) test. Overall, 13.5% of the samples from 282 infected cows had positive ELISA results, but when tested multiple times, 38.3% of the cows had at least 1 serum sample with positive results. Among 263 fecal culture-negative cows, 98.1% of the serum samples had negative ELISA results, but when tested multiple times, 7.8% of the cows had at least 1 positive ELISA sample. Fecal culture was positive on a test before the first positive ELISA in 50 cows, ELISA was positive before fecal culture in 12 cows, and in 38 cows, both tests became positive at the same testing time. An additional 174 cows were positive on fecal culture and always negative on ELISA until culled. For cows that had ELISA sample:positive (S/P) ratios below the cutoff point, the change in S/P between sequential tests was evaluated to determine whether a rise in S/P could predict infection status. In this study, change in S/P was not a useful predictor of infection status in seronegative cows.