M. paratuberculosis is the causative agent of Johne's disease, a chronic enteritis in ruminants. Simple, accurate, specific and sensitive diagnostic tests to detect clinical and subclinical disease are lacking. The mycobacterial immunodominant antigens are major stimulants of cellular and humoral immunity against pathogenic mycobacteria. The humoral immune responses of animals with Johne's disease were analyzed against M. paratuberculosis recombinant clones expressing some immunodominant antigens representing the 65K and 19K heat shock proteins (hsp65 and hsp19), the 32K secreted protein, and the specific IS900-product. The antigenic identity of these proteins was confirmed by immunoblots with specific monoclonal antibodies and/or by hybridization to specific probes. Ten cows, two goats, two sheep with clinical and five cows with subclinical Johne's disease were tested. With the exceptions of hsp65 (and one goat that reacted to recombinant clones representing the IS900), there was no reactivity seen. Three cows (33%) with clinical and 2 of 5 cows with subclinical disease cows reacted with hsp65. In addition, both sheep and neither goat reacted. We conclude that these antigens are not useful for serodiagnostics of Johne's disease.