In this study we have used saponin to permeabilize platelet membranes in order to test directly the involvement of IP3 in regulating internal Ca2+ release, and to measure IP3 binding to its receptor. Our results indicate that platelet vesicles release Ca2+ as early as 3 seconds after IP3 addition. Using [3H]IP3, we have found that platelets contain a single class of high affinity IP3 binding sites with a Kd of ∼0.20 (± 0.01) nM. Immuno-blotting shows that platelets contain a 260 kDa polypeptide which shares immunological cross reactivity with brain IP3 receptor. Immunofluorescence staining data indicate that the IP3 receptor is preferentially located at the periphery of the platelet plasma membrane. Most importantly, both IP3 binding and IP3-induced Ca2+ release activities are significantly inhibited by cytochalasin D (a microfilament inhibitor) and colchicine (a microtubule inhibitor). These findings suggest that the cytoskeleton is involved in the regulation of IP3 binding and IP3 receptor-mediated Ca2+ release during platelet activation.