Using human CD4+ T-cell clones and peptide-pulsed antigen-presenting cells (APC) we measured, at the single cell level, different steps in the T-cell activation cascade. Simultaneous analysis of T-cell antigen receptor (TCR) down-regulation and interferon-gamma (IFN-gamma) production shows that both the level of TCR occupancy and the amount of IFN-gamma produced by single T cells increase in an antigen dose-dependent fashion. Conversely, commitment of T cells to IFN-gamma production does not occur as soon as a defined number of TCR have been engaged, but requires the same duration of sustained signalling at low as well as at high antigen concentrations. Measurement of phosphotyrosine levels by flow cytometry reveals that, upon conjugation with APC, individual T cells undergo an antigen dose-dependent activation of protein tyrosine kinases (PTK), which parallels the level of TCR occupancy. In antigen-stimulated T cells the increased phosphotyrosine staining is localized in the area of contact with APC, as shown by confocal microscopy. PTK activation is sustained for at least 2 hr after conjugation, and is required to maintain a sustained increase in intracellular Ca2+ concentration. Our results show, for the first time, a direct correlation between the level of TCR occupancy and the activation of PTK in individual T cells and offer an explanation for how the number of triggered TCR can be 'counted' and integrated in a corresponding biological response.