The extracellular ionized calcium and magnesium requirements for lectin-induced lymphocyte DNA synthesis were measured in a serum-free system. The use of this system permitted measurements of the ionized calcium and magnesium concentrations with ion-selective electrodes. Maximal DNA synthesis was observed at 270 microM ionized calcium and at 100 microM ionized magnesium in phytohemagglutinin-treated lymphocytes. Lymphocyte DNA synthesis was much more sensitive to reduction of external ionized calcium than to reduction of ionized magnesium. In calcium-free medium (ionized calcium 25 microM), DNA synthesis was reduced by 90%, but in magnesium-free medium (ionized magnesium concentration 7 microM) DNA synthesis was reduced by only 30%. Fifty percent of DNA synthesis stimulated by phytohemagglutinin (PHA) and concanavalin A (Con A) was observed at external ionized calcium concentrations of 97 and 43 microM, respectively. When lymphocytes were stimulated with PHA and the external calcium was chelated with EGTA, 50% inhibition of DNA synthesis was observed at 98 microM ionized calcium. This value agreed well with the free calcium required for PHA activation of DNA synthesis (97 microM). Cytoplasmic calcium, measured with the fluorescent probe Quin 2, increased following lectin exposure if the extracellular ionized calcium concentration was greater than 80 microM. No increase in cytoplasmic calcium could be detected in lectin-treated lymphocytes below 80 microM extracellular ionized calcium, although substantial DNA synthesis was sustained.