In this report we studied the antigen identified by the 5/9 monoclonal antibody. This antigen is expressed on approximately 15% of resting T lymphocytes with helper activity and increases following T-cell activation both in vivo and in vitro. The 5/9 monoclonal antibody triggered T-cell proliferation in the presence of suboptimal doses of phorbol12-myrisate 13-acetate (PMA) but this effect was strongly inhibited by antibody-induced modulation of the CD3 T-cell-receptor complex. The observation that a number of T-cell lines were brightly stained by the 5/9 monoclonal antibody after being activated with phytohemagglutinin (PHA) and PMA allowed the molecular characterization of the 5/9 antigen as well as the analysis of the biochemical mechanisms occurring after cell stimulation with 5/9 monoclonal antibody (Mab). An activated Jurkat T-cell line was labeled with 125I on the membrane: the monoclonal antibody immunoprecipitated a molecule displaying an apparent molecular weight of 34 kDa. In addition, 5/9 molecules, purified by immunoprecipitation from Jurkat cells, were found associated to a Ca2+ phospholipid-dependent protein kinase activity.