A Map expression library in lambda ZAP was screened with mice and cattle sera to identify novel antigens. One clone was selected, sequenced and further characterized. The sequence analysis of the putative ORF predicts a protein of 20.8 kDa with a probable signal sequence compatible with Cys-acylation at Cys24, characteristic of lipoproteins. In consequence the protein was termed lpp24. We observed an important difference between the predicted (20.8 kDa) and the apparent molecular weight, either native form in Map (28 kDa) or recombinant protein in Escherichia coli (34 kDa). Upstream of lpp24 there is a sigF homologous gene. Downstream of lpp24 there is an ORF with unknown function and transcribed in the opposite sense, followed by a tetR like regulator gene. The protein was further localized in the membrane fraction of Map and extracted in the detergent phase of Triton X-114. A strong crossreaction with a protein from E. coli that has a higher size was observed. No crossreaction was observed in M. tuberculosis extract proteins. The presence of the gene in other mycobacteria was evaluated. It was observed the protein in M. avium subsp. paratuberculosis and M. avium subsp. avium and was absent in M. smegmatis, M. bovis, M. tuberculosis, M. chitae, M. africanum, M. vaccae, M. fortuitum, M. aurum, M. terrae, M. leprae, M. phlei and M. pinipedii. Humoral reactivity using bovine sera demonstrated that this protein is widely recognised by the infected and non infected animals as well, due in part to the conserved sequence in close related environmental bacteria as M. avium subsp. avium and to the presence of a conserved epitope in other bacteria as E. coli.In conclusion, these findings show that Lpp24 is a membrane protein and a putative lipoprotein present in M. avium complex and absent in M. tuberculosis complex.