Store-operated calcium entry (SOCE) is believed to contribute a major component of the thrombin-evoked increase in cytosolic Ca²⁺ concentration ([Ca²⁺]i) in human platelets. We have previously shown that SOCE, artificially induced by the sarco/endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin, is dependent on the remodelling of the actin cytoskeleton (Rosado et al. 2000). Here we have investigated the role of remodelling of the actin cytoskeleton in Ca²⁺ signalling evoked by thrombin, a physiological agonist that activates protease-activated receptors (PAR) 1 and 4 in human platelets. Fura-2-loaded human platelets were incubated at 37 °C with cytochalasin D (CytD; 10 μM) or the vehicle (DMSO) for 40 min, then stimulated by thrombin (1 U/ml), the PAR-1 agonist SFLLRN (3 μM), or the PAR-4 agonist AYPGKF (300 μM) in the presence or absence of extracellular Ca²⁺. Ca²⁺ release was estimated as the integral of the [Ca²⁺]i increase recorded for 3 min after the addition of the thrombin or AYPGKF, or 90 seconds after addition of SFLLRN, in the presence of 1 mM EGTA. Ca²⁺ entry was estimated as the integral of the Ca²⁺ response in the presence of 1 mM CaCl₂, corrected for the Ca²⁺ release over the same period. Sr²⁺ entry was used as a measure of unidirectional divalent cation flux as Sr²⁺ is reported to be a poor substrate for the PMCA, and was estimated as the integral of the 340nm/380nm fluorescence ratio over 3 min after thrombin addition in the presence of 1mM SrCl₂, corrected by subtraction of the signal due to Ca²⁺ release over the same time period. Statistical significance (p<0.5) was determined using Student's paired t tests. Inhibition of actin polymerisation with CytD increased thrombin-evoked Ca2+ entry to 148±9.2% of control (n = 12; p < 0.01). Thrombin-evoked Sr²⁺ entry was increased to 134±6.4% (n = 8; p < 0.005). Thrombin-evoked Ca²⁺ release was not significantly affected. In addition to SOCE, thrombin is reported to activate a non-capacitative pathway that is dependent on protein kinase C (PKC) (Rosado & Sage, 2000). However, inhibition of PKC with Ro-31-8220 (5 μM) did not prevent the potentiation of thrombin-evoked Ca²⁺ entry by CytD (n = 6). Neither SFLLRN-evoked Ca²⁺ release nor entry was significantly affected by CytD treatment (n = 7). AYPGKF-evoked entry was increased by 134±12.8% (n = 4; p < 0.05) by CytD, whereas AYPGKF-evoked release was not significantly affected. These data indicate that, in contrast to thapsigargin-evoked SOCE, thrombin-evoked Ca²⁺ entry is potentiated when actin polymerisation is inhibited with CytD. This effect is restricted to the PAR-4 component of thrombin signalling. CytD may prevent the internalisation of PAR-4, which is important in PAR-4 desensitisation.