We have used quantitative electron microscopic autoradiography to characterize the subcellular distribution of arachidonoyl phospholipids following brief (5 minutes) exposure of unstimulated human platelets to [3H]arachidonic acid. Labeled arachidonate was taken up rapidly and incorporated into phospholipids. Phospholipid radioactivity was preserved and spatially fixed during tissue processing for electron microscopy. Analysis of autoradiographs showed that following a brief exposure to 750 nmol/L [3H]arachidonate, there is selective labeling of an internal membrane compartment composed of the dense tubular system and the open canalicular system. The plasma membrane, platelet granules, and nonmembranous cytoplasm were not labeled. Since the open canalicular system is continuous with the plasma membrane and since phospholipids in continuous membranes are freely diffusible, our observations indicate that [3H]arachidonate was incorporated into phospholipids within the dense tubular system and not the open canalicular system. Thus, the dense tubular system, known to contain cyclooxygenase activity, incorporates arachidonate selectively following brief exposure to this fatty acid, presumably to concentrate it in proximity to enzymes for icosanoid synthesis.