This study provides a direct correlation, via dual-parameter flow cytometric analysis (simultaneous assessment of surface immunofluorescence and DNA content), between activated T-cell entry into the S/G2/M phases of the cell cycle and the kinetics of expression of a novel T-cell activation antigen, termed CK226. This molecule was identified by the specific monoclonal antibody on the leukaemic T-cell line CEM/K, and it was expressed by 8-30% of resting peripheral blood lymphocytes and the majority of monocytes and granulocytes. A large fraction of activated lymphocytes acquired the CK226 antigen before DNA synthesis. Moreover, this molecule was expressed on virtually all G0/G1 and S/G2/M phase cells by day 2 after phytohaemagglutinin (PHA) activation and at day 6 after stimulation in a mixed lymphocyte culture. The time course of expression of other known activation antigens, such as Tac and transferrin receptor, was comparable to that of CK226. Based on the relationships between CK226 expression on cycling cells and the stimulatory effects of the specific monoclonal antibody, we conclude that CK226 should be considered an early activation antigen, which defines a new pathway of T-cell activation.