The effects of the lectins concanavalin A, succinylated concanavalin A, and wheat germ agglutinin on the free cytoplasmic Ca2+ concentration in mouse thymocytes were measured using the fluorescent Ca2+ indicator "quin 2" (Tsien, R. Y. (1980) Biochemistry 19, 2396-2404) and compared with the metabolic and mitogenic effects of the lectins on the cells. Within 1 min of adding each ligand, there is a dose-dependent increase in the free cytoplasmic Ca2+ concentration reported by quin 2. This response is selective for Ca2+, but it does not coincide closely with the subsequent mitogenic stimulation at 48 h by concanavalin A or succinyl concanavalin A. The nonmitogenic lectin wheat germ agglutinin also causes an increase in free cytoplasmic Ca2+ concentration and early metabolic stimulation of the cells, but stimulation is self-aborted before DNA synthesis occurs. At the intracellular concentrations of quin 2 required for measurement of the free Ca2+ concentration, the chelator causes early metabolic stimulation of the cells very similar to that produced by concanavalin A and the mitogenic Ca2+ ionophore A23187. Thus, phosphatidylinositol metabolism and lactate production are stimulated in mouse thymocytes and pig lymphocytes within 1 h of loading with quin 2 and significant increases in RNA synthesis occur after 8 h. Quin 2 causes mitogenic stimulation of pig lymphocytes measured as increased [3H]thymidine uptake at 48 h, that is variable but substantial in most experiments (up to 100% of the stimulation by A23187). The chelator itself has no significant activity as a Ca2+ ionophore, but the apparent free Ca2+ concentration in the cells increases both with the concentration of intracellular quin 2 and with the extracellular Ca2+ concentration. These data leave open the possibility that quin 2 itself affects the concentration of free Ca2+ or other cations in the cells.