Thrombin initiates many physiological processes in platelets and other megakaryocyte-lineage cells by interacting with surface receptors and generating rises in cytoplasmic Ca2+; these rises result from both Ca2+ release from intracellular stores and receptor-mediated Ca2+ entry. Regulators that limit Ca2+ entry after its initiation by thrombin have not been identified. In this study, prevention of expression of a single protein kinase C isoenzyme (PKC beta) by antisense cDNA overexpressed in HEL cells, a human megakaryoblastic cell line that expresses thrombin receptors, promotes thrombin receptor-mediated Ca2+ entry without altering thrombin-induced intracellular release of Ca2+. The cytoplasmic Ca2+ rise initiated by endoperoxide analogs was not affected by inhibiting PKC beta. Overexpression of a cDNA encoding wild-type PKC beta mutated to prevent recognition by the antisense cDNA abolished the enhancement of Ca2+ influx following thrombin. Thus, PKC beta appears to be a specific negative regulator of thrombin receptor-mediated Ca2+ entry.