Myosin light chain₂₀ (MLC₂₀) phosphorylation is thought to be responsible for the initiation of platelet shape change. The aim of this study was to investigate the regulation of MLC₂₀ phosphorylation in platelet shape change. Previous studies have suggested that MLC₂₀ phosphorylation following platelet stimulation is dependent on intracellular calcium release. This study was designed to investigate the role of calcium sensitization in human platelet shape change and the possible intracellular mechanisms involved. An α-toxin permeabilized platelet model was developed using mock intracellular buffers to enable complete control of the intracellular environment. Permeabilized platelets were examined to ensure functional integrity by electron microscopy and were found to be capable of producing platelet shape change. Permeabilized platelets were stimulated by under calcium-clamp conditions and the degree of phosphorylation was determined following separation by two-dimensional gel electrophoresis. Calcium sensitization of platelet shape change following stimulation with both thrombin and GTPγS was produced and this corresponded with an increase in MLC₂₀ phosphorylation. A relatively slow time course was observed and this may represent a second phase of shape change as a result of secreted mediators stimulating further activation. The signalling pathway leading to calcium sensitization was investigated for the possible involvement of RhoA and Rho-kinase activation. Thrombin and GTPγS were shown to directly activate RhoA. The involvement of Rho-kinase was demonstrated using a specific inhibitor Y27632, which inhibited calcium sensitization produced as a result of thrombin and GTPγS stimulation. The calcium sensitivity of MLC₂₀ phosphorylation in platelet shape change is increased by thrombin stimulation and this is likely to involve RhoA and Rho-kinase activation. This is not calcium-independent regulation but does have a requirement for submaximal intracellular calcium concentration to produce an increase in MLC₂₀ phosphorylation. This alternative pathway which is not dependent on intracellular calcium release may provide an alternative target in the treatment of disease states associated with haemostasis.