45Ca 2+ uptake was detected within minutes following addition of T- and B-cell 2 2 Abbreviations used: T cells, thymus-dependent T lymphocytes; B cells, thymus-independent B lymphocytes; MEM, modified alpha minimum essential serum-free medium (containing 1.8 m M Ca 2+ and 2 m M l-glutamine), buffered with 20 m M Hepes (instead of HCO 3 −) to pH 7.2, containing penicillin G (100 units/ml) and streptomycin (100 μg/ml); cpm, counts/min; Con A, 3 × crystallized concanavalin A; PHA, purified P. vulgaris phytohemagglutinin; LPS, bacterial lipopolysaccharide; A23187, a divalent Ca 2+ ionophore; Gln, l-glutamine; AIB, α-aminoisobutyric acid; PBS, phosphate-buffered saline, pH 7.2; db cAMP, sodium salt of N 6,O 2 -dibutyryl adenosine 3′,5′-cyclic monophosphoric acid; db cGMP, sodium salt in N 2,O 2 -dibutyryl guanosine 3′,5′-cyclic monophosphoric acid; 8-Br cGMP, 8-bromoguanosine 3′,5′-cyclic monophosphoric acid; PG, prostaglandin; FCS, heat-inactivated fetal calf serum; NWSM, water-soluble mitogen of Nocardia opaca; DMSO, dimethylsulfoxide. mitogens to mouse lymphocytes. The T-cell mitogens (Con A and PHA) gave an ~twofold increase in 45Ca 2+ uptake (representing an influx of ~ 130 amol per lymphocyte, corresponding to an increase in average cellular Ca 2+ of ~0.95 m M). B-cell mitogens which gave the largest 45Ca 2+ uptake (~twofold) were purified LPS preparations from Salmonella minnesota R595 and Escherichia coli 0111:2125. The 45Ca 2+ uptake by rabbit splenocytes using specific anti-b4 allotype antiserum was comparable to that obtained with the two purified LPS preparations. A23187, in low nontoxic doses, gave an ~sixfold increase in 45Ca 2+ uptake with mouse T cells. The 45Ca 2+ uptake was modulated by cyclic nucleotides showing a “yin-yang” effect. The results suggest a possible entry of 45Ca 2+ from the extracellular medium through “gated Ca 2+ channels” in the plasma membrane into the cytosol by passive diffusion. The Ca 2+ may be sequestered in the mitochondria, and the excess Ca 2+ is later effluxed into the extracellular medium. The fact that 45Ca 2+ uptake appears to be one of the earliest events occurring after ligand binding to the cell, together with the demonstration of a Ca 2+-dependent glucose uptake and a requirement for extracellular Ca 2+ for DNA synthesis, suggest that, as it is now known to function in many other cellular responses, Ca 2+ may operate as a second messenger for lymphocyte activation.