The study of the interaction of the immune system with molecules from pathogens may lead valuable information of mechanisms involved during infection and potential tools for the diagnosis. The aim of the present study was to evaluate an expression library of Mycobacterium avium subsp. paratuberculosis (Mptb) constructed in lambda zap II to identify new antigens. Sera from naturally infected bovines or from mice inoculated with the whole bacteria or sonicated was used for the immunoscreening of the library. Sera were previously analysed by Western blot to determine the number and strength of band recognition. Bacterioferritin (antigen D) was identified with the bovine pooled sera. From the analysis of the library with mice serum several antigens were identified, some of them already described as antigens in Mptb and other mycobacteria: HSP70 and dnaK. Other potentially novel antigens were identified by sequence similarity with M. tuberculosis genome as serine/threonine protein kinase (99% of identities with pknB from M. tuberculosis, Rv 0014), isocitrate lyase (aceAa, 100% of identity with Rv 1915). In other hand, a sequence of 876bp which codes for a peptide of 32kDa was present in M. avium subsp. avium (99% of identity), but absent in the genome of M. tuberculosis. No previous description of this sequence was found (BLAST). The recombinant antigens fraction was recognised by all the bovine infected sera when were assayed by Western blot.