Paratuberculosis (PTB) is characterized by chronic enteritis as a consequence of Mycobacterium avium paratuberculosis (Map)infection in cattle. Map shares several antigens with other Mycobacteria, including M. bovis. Some attempts with purified antigens have been described in order to differentiate between those two infections in cattle. The purpose of this study was to demonstrate the potential of two M. bovis-specific recombinant proteins MPB70 or MPB83 to distinguish PTB from bovine tuberculosis infection (TB).Two TB-free and six herds where TB occurs were studied. An ELISA using the recombinant proteins MPB70 and MPB83 was standardized. Two distinct populations of animals were selected, Group A (n = 23) with animals coming from TB-free herds with PTB and Group B (n = 48) composed by PTB-free animals (confirmed by culture) from where M. bovis infection was cultured. In Group A, 10 animals reacted to MPB70 (43.47%) and nine to MPB83 (39.13%). In Group B, 37 animals reacted to both MPB70 and MPB83 (77.08%). Our results indicate that both antigens presented very similar results, with a concordance (kappa index) of 0.91. The difference between the mean OD value for Group A and Group B measured against the same antigen was highly significant (p<0.01). MPB70 and MPB83 clearly detect M. bovis specific antibodies more often in tuberculous cows than in paratuberculous cattle, and therefore can be considered as valuable tools to differentiate between these two infections at serology. The use of recombinant antigens derived from each microorganism, or a combination of them, would increase the specificity of serological assays by diminishing antigenic cross-reactivity between bovine PTB and TB.