Prostaglandin type El (PGEl) rapidly stimulates cyclic AMP formation and the initiation of deoxyribonucleic acid (DNA) synthesis in rat thymic lymphocytes suspended in vitro by reactions which are not affected by wide variations in the extracellular calcium concentration. On the other hand, the operation of the associated reaction(s) responsible for the subsequent progression of the stimulated cells into mitosis is profoundly affected by the extracellular calcium level. If the maximum intracellular cyclic AMP concentration is in the lower range of stimulatory values (e.g., 150 X picomoles per cell as produced by a n exposure to 0.5 p g of PGEl per milliliter of medium), an extracellular calcium concentration of 0.5 to 1.0 miv is needed to obtain niaximum cell proliferation, but not the maximum stimulation of DNA synthesis. Contrariwise, if the cellular cyclic AMP content is raised to a much higher level (260 X 10-8 picomoles per cell) by exposure to a greater PGEl concentration (5.0 yg per milliliter), cell proliferation is maximally stimulated in calcium-free medium and increasing the extracellular calcium concentration above 0.2 mM actually prevents the stimulation of cell proliferation (but does not affect the stimulation of DNA synthesis). Thus, the ultimate translation of PGEl's early cyclic AMP-mediated reactions into increased cell proliferation is determined by both the intracellular cyclic AMP level and the extracellular calcium concentration.