T lymphocytes isolated from mice infected with Mycobacterium tuberculosis response vigorously to proteins secreted by the bacilli and these antigens may be of importance in the generation of protective immunity against the disease. In this study, short-term culture filtrate (ST-CF), which constitutes a complex mixture of secreted proteins, was fractionated by a modified preparative SDS-PAGE technique. The ability of each fraction to be recognized by T cells isolated from infected mice was evaluated by quantifying proliferation and IFN-gamma production in cell cultures. Two molecular mass regions 4-11 and 26-35 kDa were found to possess marked stimulatory properties. Four potent single antigens were mapped within the stimulatory regions. These purified antigens stimulated T cells isolated from mice at the height of a tuberculous infection to produce large amounts of IFN-gamma. Two of these stimulatory antigens belonged to the antigen 85 complex.