A novel Mycobacterium bovis antigen was identified from an expression library using sera from naturally infected cattle. The Escherichia coli recombinant clone expressed a 27 kDa protein, named P27. A rabbit serum against the recombinant antigen recognized a protein of 27 kDa in cellular extracts from M. bovis and M. tuberculosis. No protein was recognized in the culture supernatant. Sequence analysis indicated that P27 has a molecular mass of 24 kDa, showing a characteristic signal sequence for lipoprotein modification (a signal peptidase type II site). The gene is identical to a gene identified in the M. tuberculosis genome sequencing project. Cellular fractionation experiments suggested that P27 is an integral membrane protein. The antigen was recognized by individual sera and peripheral blood mononuclear cells (PBMC) from diseased cattle. PCR experiments with specific primers directed to the P27 structural gene indicated that it is only present in the M. tuberculosis species complex. In conclusion, a novel immunogenic lipoprotein in M. bovis/M. tuberculosis has been identified. The results presented here and elsewhere suggest that mycobacterial lipoproteins should be considered in the design of new recombinant vaccines and diagnostic methods.