We have investigated Ca2+ mobilization in single T cells stimulated with their physiological ligand, i.e. antigenic peptide bound to major histocompatibility complex (MHC) molecules on antigen-presenting cells (APC). Fibroblasts expressing I-Ed class II molecules were pulsed with a peptide derived from the lambda 2(315) immunoglobulin light chain. Onto such antigen-pulsed fibroblasts were sedimented cloned Th1 cells loaded with Fura-2. Changes in cytosolic Ca2+ concentration in single T cells were continually monitored by use of an imaging system based on fluorometry. Ca2+ mobilization was both peptide-specific and MHC-restricted. Within seconds of the initial APC-T cell contact, a Ca2+ spike could be observed. The Ca2+ response gradually declined over a 25-min period, during which oscillations were noted. Various parameters characterizing the magnitude of the Ca2+ response (latency, increase rate, max and mean Ca2+ increase, frequency and period of oscillations) all correlated with the amount of peptide used for pulsing the fibroblasts. Thus, Ca2+ mobilization in single T cells appears not to be an all or none phenomenon. Rather, activation is incremental (analog signaling), the degree of Ca2+ mobilization probably being related to the number of stimulatory peptide-MHC complexes on the surface of the APC. The extent of calcium mobilization and lymphokine production (interleukin (IL)-2, IL-3, interferon-gamma) correlated, at least at the population level.