Thrombin responses in human platelets are mediated by the protease-activated receptors (PAR), PAR1 and PAR4. The signalling pathways mediating PAR activation have not been fully delineated for human platelets. We assessed cytoplasmic Ca2+ mobilization in response to activation with thrombin and PAR1 (SFLLRN) and PAR4 (GYPGKF) peptides in two patients whose platelets were deficient in two major signalling proteins, Galphaq or phospholipase (PLC)-beta2. In normal platelets, thrombin induced a biphasic Ca2+ response with a rapid rise to a peak followed by a sustained elevation in Ca2+. The peak Ca2+ rise was impaired in both patients at lower thrombin concentrations. At higher concentrations, it was decreased in PLC-beta2-deficient platelets; the sustained Ca2+ elevation observed in normal and Galphaq-deficient platelets was reduced in PLC-beta2-deficient platelets. The response to SFLLRN was decreased in both patients at lower concentrations. The peak Ca2+ in response to GYPGKF was reduced in both patients; the sustained Ca2+ increase was markedly decreased in PLC-beta2-deficient platelets. These studies provide evidence that, in human platelets, both Galphaq and PLC-beta2 play a major role in responses to PAR1 and PAR4 activation, and that PLC-beta2 is required for the sustained Ca2+ rise upon thrombin activation.