This report describes extraction of a new native antigen fraction from Mycobacterium tuberculosis without massive degradation of proteins by Triton X-100. The Triton X-100 solubilised protein (TSP) antigen showed a characteristic antigen profile and reproducible extraction pattern. To characterise the nature of their composition, the TSP antigen was fractionated by Triton X-114 phase partitioning. The TSP antigen contained a variety of lipids and glycoconjugates as well as diverse proteins. Most proteins were partitioned into the aqueous phase during phase fractionation, whereas non-protein molecules and lipoproteins were recovered in the detergent phase. The lymphoproliferative responses to the TSP aqueous fraction in healthy tuberculin reactors were significantly higher than those to the purified protein derivative (PPD) and unfractionated TSP. In contrast, the antibody responses to TSP aqueous fraction in tuberculosis patients showed weak reactivity. This study suggests that the TSP aqueous fraction can be used as a T-cell antigen associated with protective immunity against tuberculosis.