An increase in intracellular calcium concentration ([Ca²⁺]_i) is an important step in the activation of human platelets. Activation of protease-activated receptors (PARs) 1 and 4 by thrombin evokes release of Ca²⁺ from intracellular Ca²⁺ stores and Ca²⁺ influx, and also increases protein kinase C (PKC) activity in platelets. PKC regulates both Ca²⁺ entry (Rosado & Sage, 2000) and removal of Ca²⁺ from the cytosol. We have investigated the role of PKC in regulation of thrombin-evoked Ca²⁺ release. [Ca²⁺]_i was recorded from fura-2-loaded platelets at 37°C. Statistical significance was analysed using a paired t test; p<0.05 was considered significant. Ro-31-8220 (5 μM, 5 min), which inhibits both conventional (Ca²⁺-dependent) and novel (Ca²⁺-independent) PKC isoforms, reduced the elevation in [Ca²⁺]_i evoked by thrombin (1 unit/ml) in the presence of 1 mM CaCl₂ to 67 ± 9 % of control (mean ± S.E.M.; n = 6; p<0.05). Ca²⁺ release (in the presence of 1 mM EGTA) was increased to 122 ± 7 % of control (n = 6; p<0.05), which may reflect regulation of the plasma membrane Ca²⁺ ATPase by PKC. The early spike in Ca²⁺ release, which has been attributed to activation of PAR-1 was abolished, indicating that PKC regulates thrombin-evoked Ca²⁺ release. SFLLRN (30 μM), a PAR-1 agonist, stimulated Ca²⁺ release from intracellular Ca²⁺ stores. Ro-31-8220 reduced the maximum elevation of this release to 69.5 ± 4.0 % of control (n = 7; p < 0.005). In contrast, Gö6976, which selectively inhibits conventional PKC isoforms, had no significant effect on the Ca²⁺ release (n = 6), suggesting that activation of a novel PKC isoform may have a positive regulatory role in PAR-1-evoked Ca²⁺ release. Cyclosporine A (CSA), which inhibits the type 2B serine/threonine phosphatase, calcineurin, is known to also induce platelet hyperaggegability. CSA (10 μM, 10 min) significantly enhanced the SFLLRN-evoked maximum elevation in [Ca²⁺]_i to 126.7 ± 5.6 % (n = 12; p < 0.001). However, CSA had no effect on the Ca²⁺ release in cells that had also been treated with Ro-31-8220 (n = 7), suggesting that Ca²⁺ release is reciprocally regulated by PKC and calcineurin. In conclusion, PAR-1-dependent Ca²⁺ release is enhanced by PKC and inhibited by calcineurin. Calcineurin may modulate changes in [Ca²⁺]_i, and thus platelet activation, by regulating the level of PKC-dependent phosphorylation of proteins involved in Ca²⁺ signalling. A possible candidate protein for this regulation is the inositol-1,4,5-trisphosphate receptor. This PKC-dependent increase in Ca²⁺ release may lead to an increased store-operated Ca²⁺ entry and enhanced platelet activation.