G proteins play a major role in signal transduction upon platelet activation. We have previously reported a patient with impaired agonist-induced aggregation, secretion, arachidonate release, and Ca²⁺ mobilization. Present studies demonstrated that platelet phospholipase A₂ (cytosolic and membrane) activity in the patient was normal. Receptor-mediated activation of glycoprotein (GP) IIb-IIIa complex measured by flow cytometry using antibody PAC-1 was diminished despite normal amounts of GPIIb-IIIa on platelets. Ca²⁺ release induced by guanosine 5′-[γ-thio]triphosphate (GTP[γS]) was diminished in the patient’s platelets, suggesting a defect distal to agonist receptors. GTPase activity (a function of α-subunit) in platelet membranes was normal in resting state but was diminished compared with normal subjects on stimulation with thrombin, platelet-activating factor, or the thromboxane A₂ analog U46619. Binding of ³⁵S-labeled GTP[γS] to platelet membranes was decreased under both basal and thrombin-stimulated states. Iloprost (a stable prostaglandin I₂ analog) -induced rise in cAMP (mediated by Gα_s) and its inhibition (mediated by Gα_i) by thrombin in the patient’s platelet membranes were normal. Immunoblot analysis of Gα subunits in the patient’s platelet membranes showed a decrease in Gα_q (<50%) but not Gα_i, Gα_z, Gα₁₂, and Gα₁₃. These studies provide evidence for a hitherto undescribed defect in human platelet G-protein α-subunit function leading to impaired platelet responses, and they provide further evidence for a major role of Gα_q in thrombin-induced responses.