Western blots of sera from calves infected with Mycobacterium paratuberculosis demonstrated an early antibody response that has potential diagnostic applications. Antibody responses in 13 calves at 6, 12, 18 and 24 months of age and of known M. paratuberculosis infection status were investigated. These included 4 uninfected calves born to test negative dams obtained from farms with a low prevalence of Johne's disease, 3 naturally infected calves born and raised by cows shedding M. paratuberculosis in their faeces and 6 artificially infected calves which were either orally dosed with a field isolate of M. paratuberculosis or dosed with intestinal mucosa from a clinically infected cow. A sonicated cell lysate of M. paratuberculosis was separated by 10% SDS-PAGE and transferred to nitrocelluase membrane for Western blotting. Antibody reactions to the separated antigens were detected using anti-bovine IgG (whole molecule) alkaline phosphatase conjugate. Multiple reactions to antigen bands within a molecular weight range of 12 kDa to 162 kDa were observed in the sera of infected and uninfected calves. Each calf reacted to between 4 and 13 antigen bands. Bands which were specific and only recognised by serum antibodies of infected calves increased in number over time from 7 bands at 6 months, to 16 bands at 12 months, 35 bands at 18 months and 25 bands at 24 months. The number of non-specific antigen bands (common to sera of both test groups as well as unique to sera of uninfected calves) totalled 57% of all bands observed on the blots and did not increase over time.