OBJECTIVE: To investigate the diagnostic potential of four Mycobacterium tuberculosis antigens encoded by M. tuberculosis-specific region of difference 1 (RD1) region genes (PE35, PPE68, culture filtrate protein 10 [CFP-10], early secreted antigenic target-6 [ESAT-6]) and RD9 region gene Rv3619c, for delayed-type hypersensitivity (DTH) responses in guinea pigs. DESIGN: Recombinant M. tuberculosis proteins were expressed in Escherichia coli and purified to homogeneity by affinity chromatography. Guinea pigs were injected with heat-killed M. tuberculosis and live bacille Calmette-Guérin (BCG), M. avium and M. vaccae. Two to four weeks later, the guinea pigs were challenged intradermally in the flank region with mycobacterial sonicates and purified recombinant proteins. The DTH responses were quantitated by measuring erythema at injection sites after 24 h. RESULTS: All mycobacterial sonicates induced positive DTH responses in guinea pigs injected with M. tuberculosis, M. bovis BCG, M. avium and M. vaccae. Purified proteins PE35, PPE68, CFP10 and ESAT-6 elicited positive DTH responses in the M. tuberculosis-injected group but not in BCG-, M. avium- and M. vaccae-injected guinea pigs, whereas Rv3619c elicited positive DTH responses in the M. tuberculosis- and BCG-injected groups, but not in the M. avium- and M. vaccae-injected guinea pigs. CONCLUSION: The recombinant RD1 antigens induced M. tuberculosis-specific DTH responses. These antigens may therefore be useful in the diagnosis of tuberculosis.