Upon stimulation with physiological agonists, calcium ionophores or under shear stress, platelets, which express, among others, caspases 3 and 9, might develop apoptotic events. Caspase 3 and 9 activation and translocation were detected by caspase activity assay and Western blotting using specific antibodies. Platelet treatment with thrombin results in activation of caspases 3 and 9 and significantly increases the amount in the cytoskeleton of the active forms of both caspases and the procaspases 3 and 9. Pretreatment of platelets for 10 min with 5 mM Ro-31-8220, a PKC inhibitor, significantly reduced thrombin-induced caspase 3 and 9 activation and translocation, which demonstrates that caspases activation and association with the cytoskeleton needs the contribution of PKC. Finally, our results show that inhibition of thrombin-induced caspase activation has no effect on their translocation to the cytoskeleton although impairment of thrombin-evoked caspase translocation has negative effects on caspase activity, suggesting that translocation to the cytoskeleton might be important for caspase activation by thrombin in human platelets. Supported by MEC-DGI grant BFU2004-00165 and Ministerio de Asuntos Exteriores y Cooperación de España (38/04/P/E).