Abstract Varying the extracellular calcium concentration between 0 and 1.0 mM does not affect the proliferation of rat thymocytes maintained in vitro. However, higher calcium concentrations raise the level of cell proliferation and increase the intracellular concentration of cyclic AMP. Calcium's mitogenic action is inhibited by imidazole, a compound which increases the activity of the cyclic AMP-degrading enzyme phosphodiesterase and thereby also prevents calcium from increasing the cellular cyclic AMP content. Caffeine, which inhibits phosphodiesterase activity, sensitizes thymocytes to the mitogenic action of calcium. Caffeine can also stimulate thymocyte proliferation by itself and, like calcium, causes a significant rise in the cellular cyclic AMP content. Mitogenic levels of calcium, in turn, act like caffeine and reduce the activity of cyclic AMP-phosphodiesterase. These observations combine to suggest that calcium is the physiological equivalent of caffeine and exerts its effect on thymocyte proliferation by reducing the activity of cyclic AMP-phosphodiesterase.