Evidence that Ca2+ may serve as a physiologic regulator of post-transcriptional protein synthesis was recently reported (Brostrom, C. O., Bocckino, S. B., Brostrom, M. A., and Galuska, E. M. (1986) Mol. Pharmacol. 29, 104-111). To evaluate further the role of Ca2+ in translation, the polysomal contents of Ca2+-depleted and -restored GH3 pituitary cells were compared. Ca2+ depletion of intact cells with 1 mM EGTA resulted in the disappearance of polysomes and an accumulation of 80 S monosomes and ribosomal subunits typical of slowed rates of initiation. Ca2+ repletion rapidly (minutes) restored cellular polysomal contents with an accompanying accumulation of 43 S preinitiation complex (40 S.eukaryotic initiation factor 2.Met-tRNAf.GTP). Comparable polysomal profiles were found for Ca2+-depleted and -restored cells exposed to cycloheximide which apparently slowed polypeptide chain elongation to rate-limiting values in the overall translation process. Ribosomal transit times for both Ca2+-depleted and -restored cells were identical, indicating that elongation is not directly affected by the cation. Transit times were extended in parallel as a function of increasing cycloheximide concentration. Lysates of GH3 cells exhibited incorporation that was proportional to the polysomal contents derived from the original intact cell preparations. Such lysates did not possess the ability to initiate new peptide synthesis and were not affected by Ca2+ or EGTA. Ca2+-depleted cells exposed to cycloheximide provided lysates with identical elongation activity to that of lysates prepared from either comparably treated or control Ca2+-restored cells. Ca2+ is proposed to regulate translation in intact cells through modulation of the rate of initiation rather than either polypeptide chain elongation or termination.