The use of defined protein and peptide antigens can overcome specificity limitations of purified protein derivatives in the detection of bovine tuberculosis when the antigens are used in blood-based tests. Since the use of these specific antigens as skin test reagents could have practical advantages, we investigated the potential of Mycobacterium bovis-specific antigens to stimulate delayed-type hypersensitivity (DTH) responses in cattle experimentally infected with M. bovis. A cocktail of the recombinant antigens ESAT-6, MPB83, and MPB64 failed to stimulate in vivo DTH in cattle that had been experimentally infected with M. bovis despite the fact that the antigens were recognized in vitro by the same animals. However, it was possible to stimulate antigen-specific bovine DTH responses by using ESAT-6 in combination with a synthetic bacterial lipopeptide. This lipopeptide stimulated the release of the proinflammatory cytokine tumor necrosis factor alpha from monocyte-derived bovine dendritic cells in vitro, thereby providing a possible mechanism for its DTH-enhancing properties.