In this study, we characterised the mechanisms of Rac GTPase activation in human platelets stimulated by two physiological agonists, either thrombin, acting through membrane receptors coupled to heterotrimeric G-proteins, or collagen which is known to mobilise a tyrosine kinase-dependent pathway. Both agonists induced a rapid activation of Rac that was not significantly affected by the inhibition of integrin alpha(IIb)beta(3) engagement. Using pharmacological inhibitors, we found that phospholipase C activation and calcium mobilisation were essential for platelet Rac activation by either thrombin or collagen whereas protein kinase C inhibition was without effect. In contrast to Rac, Cdc42 activation was independent of phospholipase C activation, indicating that the two GTPases are differently regulated. We also found that phosphoinositide 3-kinase was not required for Rac activation in response to thrombin but was involved in its activation by collagen.