Ca²⁺ entry in human platelets can be activated by depletion of intracellular Ca²⁺ stores. This store-operated Ca²⁺ entry (SOCE) is reduced if remodelling of the actin cytoskeleton is inhibited (Rosado et al., 2000), suggesting platelet SOCE is activated by secretion-like coupling, where trafficking of endoplasmic reticulum towards the plasma membrane allows coupling between proteins in these two membranes. Platelet Ca²⁺ store depletion results in de novo coupling of the type II inositol 1,4,5-trisphosphate receptor (IP₃RII) to the Ca²⁺ entry channel hTRPC1 (Rosado & Sage, 2000). Coupling is reduced if cytoskeletal remodelling is inhibited. An essential requirement of the secretion-like coupling hypothesis is that remodelling of the cortical F-actin layer occurs rapidly enough to allow membrane trafficking and protein coupling to account for the observed Ca²⁺ entry. Here we have used a quenched-flow approach to examine thrombin-evoked changes in platelet F-actin content on a sub-second timescale. The latency of thrombin-evoked Ca²⁺ release was determined using stopped-flow fluorimetry with fura-2-loaded platelets (Brownlow & Sage, 2003). For determination of platelet F-actin content, cells were mixed with thrombin (or buffer as control) for various time intervals before fixing with 3% (w/v) paraformaldehyde in phosphate buffered saline using a rapid quench flow system. Platelet F-actin content was then determined as previously described (Rosado et al., 2000). All experiments were conducted at 37 °C. Thrombin (0.1 unit ml^-1) evoked Ca²⁺ release from intracellular stores (in the presence of 1 mM EGTA) with a delay of 1.42 ± 0.15 s (mean ± S.E.M., n = 15). Thrombin (0.1 unit ml^-1) evoked an initial decrease in platelet F-actin that commenced within 0.1 s and reached a minimum 1 s after stimulation. The F-actin content then increased, exceeding basal levels again approximately 3 s after stimulation. Mixing cells with agonist free buffer did not evoke significant changes in F-actin content. Our results indicate that thrombin stimulates rapid actin depolymerisation in platelets before F-actin increases above resting levels. The actin depolymersation precedes release of Ca²⁺ from intracellular stores, indicating that it occurs via a Ca²⁺-independent pathway. Rapid actin depolymerisation is compatible with membrane trafficking underlying the de novo coupling of IP₃RII to hTRPC1, which occurs with minimal temporal separation from thrombin-evoked Ca²⁺ release and Ca²⁺ entry (Brownlow & Sage, 2003).