Three serum ELISAs for detection of antibodies against Mycobacterium avium subsp. paratuberculosis (M. ptb.) were evaluated against culture of tissue and feces samples from 994 dairy cows collected at slaughter between January and October 2001. Culture of ileum and associated lymph nodes was performed using the Trek ESP® Culture System II. Overall, cultures for M. ptb. were positive in 160 (16.1%) of the 994 cows and 36 (3.6%) were fecal culture-positive for M. ptb. Two of the ELISAs evaluated were absorbed indirect assays and the third was a non-absorbed indirect assay. Estimated sensitivities of the absorbed ELISAs when compared to tissue culture were 8.8% (95% CI: 4.4 - 13.1%) and 6.9 % (3.0 - 10.8%), while the unabsorbed ELISA has a sensitivity of 16.9% (11.0 - 22.7%). Specificities were 97.6% (96.6 - 98.6%), 96.0% (94.7 - 97.4%) and 90.8% (88.8 - 92.7%) respectively. When compared to fecal culture, the sensitivities of the absorbed ELISAs were 16.6% (4.6 - 28.8%) and 13.9% (2.6 - 25.2%), respectively, and the sensitivity of the unabsorbed ELISA was 27.8% (13.1 - 42.4%). Specificities were 97.1% (96.0 - 98.1%), 95.9% (94.6 - 97.2%) and 90.1% (88.2 - 92.0%), respectively. Measurement of test accuracy was performed using receiver operator characteristic curves, and the area under the curves (AUC) for the absorbed ELISAs when tissue culture was the standard were 0.553 and 0.547, while the unabsorbed ELISA had an AUC of 0.540. When fecal culture was the comparison standard, the AUC of the absorbed ELISAs was 0.575 and 0.574, while the unabsorbed ELISA was 0.529. Overall, the sensitivities of the ELISAs when compared to tissue culture were low. The apparent advantage of the unabsorbed ELISA with respect to sensitivity is at the cost of lowered specificity and test accuracy.