A B-cell epitope on the carboxy-terminal region of the mycobacterial 65-kDa heat shock protein that distinguishes Mycobacterium tuberculosis/Mycobacterium bovis BCG from Mycobacterium leprae was identified by two novel monoclonal antibodies (mAbs), Ne5 and Nd4. These mAbs also showed a limited cross reactivity with mycobacterial species belonging to M. tuberculosis complex and Mycobacterium avium complex with the exception of Mycobacterium vaccae. Characterization of the epitope recognized by these mAbs was done with M. bovis BCG 65-kDa fusion proteins expressed in Escherichia coli encoding various segments of the 65-kDa protein. Our results together with those reported in literature indicated that this epitope resides in the highly divergent region of amino acid residues 525 to 540. This B-cell epitope on the 65-kDa protein of M. tuberculosis/M. bovis BCG has not been recognized by previously reported mAbs, although the analogous epitope sequence of M. leprae 65-kDa has been identified by a known mAb (IIIC8) reported in the literature. Therefore Ne5/Nd4 epitope could be considered important in studying the differential immune response of the host against infections with M. tuberculosis complex/M. avium complex and M. leprae.