To ascertain possible interactions of intracellular Mg2+ and Ca2+, the concentration of intracellular free Ca2+ ([Ca2+]i) and intracellular free Mg2+ ([Mg2+]i) were experimentally increased within physiological ranges and it was measured by means of fura-2 and mag-fura-2 whether the increased concentration of one divalent cation affected the concentration of the other. A four-fold increase of [Ca2+]i in rat thymocytes by concanavalin A or thapsigargin had no significant effect on [Mg2+]i. Incubation of rat thymocytes in Na+-free medium increased [Mg2+]i from 0.35 to 0.7 mM. This increase in [Mg2+]i did not affect [Ca2+]i or the action of thapsigargin on [Ca2+]i.