The aim of this study was to identify possible immuno-dominant and M. avium spp. paratuberculosis-specific antigenic components in ovine paratuberculosis by analysing large numbers of sheep sera by immunoblot. Sera were used with and without pre-absorption with a M. phlei sonicate. The sera comprised serial bleeds from 44 sheep, experimentally infected with M. paratuberculosis, sera from lesion-confirmed, naturally M. paratuberculosis-infected sheep, and three sera groups from sheep free from paratuberculosis, namely 355 sera from Australia and 156 sera from the Falkland Islands. All sera were also tested in an absorbed ELISA. With sera from experimentally infected sheep protein bands of 47, 37, 30, 24 and 21 kDa and a broad band of a polysaccharide of 32 to 42 kDa were detected. A few other bands appeared infrequently, either of low molecular weight below 21 kDa or of high molecular weight around 100 kDa. Altogether, sera from 37 sheep stained bands in blots during the course of infection with variable frequencies. Sera from naturally infected, lesion-confirmed sheep stained the same bands as observed with the experimental sera. Some staining of bands was also seen with negative sera but most of these bands could be absorbed out. The results of this study indicate that the 32-42 kDa polysaccharide is immunodominant and shows a certain degree of specificity, while protein antigens appear to be less specific.