An agar gel immunodiffusion test (AGID) utilizing a commercially available antigen, was used to detect M. paratuberculosis-infected animals in a field study. Over 2000 serum samples obtained from 5 infected and 4 presumed-uninfected sheep flocks were tested over a 5-year period. A total of 35 AGID-positive sheep were identified and 27 of these were available for necropsy. Infection was confirmed in all necropsied sheep by histopathology, acid-fast staining of ileal mucosal smears, and/or bacterial culture. Histopathology was a reliable method of confirming paratuberculosis and demonstrated a wide spectrum of lesions. Isolation of M. paratuberculosis by culture of feces and tissues of infected animals was successful in only three cases. The AGID was useful in identifying infected sheep with weight loss. In those animals which were repeatedly tested, AGID appeared to identify most infected animals late in the course of disease progression, however, ten animals were in normal body condition when necropsied or lost to follow-up (one animal). Exposure of animals to or infection with Corynebacterium pseudotuberculosis did not appear to cause false positive reactions in the AGID. It was concluded that AGID with the commercially available antigen may be useful to identify M. paratuberculosis-infected sheep with weight loss, and that it may be useful in flock testing programs.