The subcellular distribution of the alpha 2-adrenergic receptor, pertussis-toxin substrates (Gi, the inhibitory G-protein) and adenylate cyclase was determined in human platelets. The alpha 2-adrenergic receptor and pertussis-toxin substrate activity codistribute with surface membranes identified by a novel fluorescent-lectin method. The platelet granule fractions did not contain detectable Gi. Only 2-4% of the total pertussis-toxin substrate activity appears in soluble fractions, and this amount was not increased upon addition of purified beta gamma units or after pretreatment of platelets with adrenaline. There is no evidence for compartmentation of the alpha 2-adrenergic receptor or Gi to account for the low-affinity component of agonist binding to the alpha 2-adrenergic receptor in human platelet membranes. Translocation of Gi from plasma membrane to platelet cytosol or granules does not appear to play any significant role in the mechanism of alpha 2-receptor-mediated platelet activation.