Paratuberculosis (Johne's disease), is a chronic enteritis caused by Mycobacterium paratuberculosis that affects a large proportion of ruminants world-wide and is responsible for enormous economic losses. Currently available serologic tests have poor sensitivity and specificity. We previously described the construction of an expression library of M. paratuberculosis in E. coli. In this study, we used the gene product of a recombinant clone expressing a 35KD (p35) of M. paratuberculosis recombinant antigen for serodiagnosis of Johne's disease. The seroreactivity of p35 was evaluated by immunoblotting against sera of 57 reference animals. P35 was recognized by sera from all 12 cattle, 2 goats and 2 sheep (100%) with advanced Johne's disease (Clinical Stage) and by 15 of 20 (75%) cattle with early infection (Subclinical Stage). None, of the 15 M. paratuberculosis free cows, 3 BCG-infected cows (Tuberculous cattle) or the 3 cows, artificially inoculated with multiple doses of viable M. paratuberculosis, reacted with p35. The overall sensitivity, specificity, positive predictive value and negative predicted value were 86%, 100%, 100%, and 75%, respectively. These data are much higher than those reported using commercially available diagnostic tests. In addition, p35 reacted poorly to sera from tuberculosis, leprosy and Crohn's disease patients. When the DNA fragment encoding p35 gene was used as a probe, it hybridized to DNA only from M. avium complex but not to DNA from any other mycobacteria or non-mycobacterial organisms that were tested. Our data clearly suggest that p35 recombinant protein and p35 encoding gene DNA fragment have potential for efficient serodiagnosis of animals with Johne's disease at all stages of the infection and as a probe for identification of M. avium complex infection, respectively.