Thrombin-induced activation of RhoA and its involvement in the regulation of myosin II light chain(20) phosphorylation (MLC-P) in alpha -toxin permeabilized platelets was investigated. Permeabilized platelets, expressing normal levels of P-selectin, displayed a Ca2+-dependent increase in shape change and MLC-P. Thrombin activated RhoA as measured by a rhotekin-binding assay within 30 s of stimulation under conditions of constant [Ca2+](i). Under the same conditions and timecourse, thrombin or GTP gammaS induced an increase in MLC-P and platelet shape change which was not dependent on an increase in [Ca2+](i). The thrombin- and GTP gammaS-induced MLC-P in constant [Ca2+](i) was inhibited by the addition of Y27632, a Rho-kinase inhibitor. This study directly demonstrates that thrombin can activate RhoA in platelets in a timecourse compatible with a role in increasing MLC-P and shape change (not involving an increase in [Ca2+](i)). This is also Rho-kinase-dependent.