SUMMARY.The role of intracellular histamine in the activation of human platelets was explored using the novel histamine antagonist N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine (DPPE). DPPE inhibited aggregation, [32P]-phosphatidic acid production (an index of phospholipase C activity) and the increase in cytosolic calcium ([Ca2+]i) in response to collagen. In contrast, while at higher concentrations DPPE inhibited aggregation in response to ADP and the thromboxane mimetic EP171, it failed to inhibit EP171-induced [32P]-phosphatidic acid production or increases in [Ca2+]i elicted by either ADP or EP171. Collagen but neither ADP nor EP171 caused the significant formation of intracellular histamine. The biochemical changes induced by collagen have been shown previously to be, at least partly, secondary to thromboxane generation. Collagen-induced arachidonic acid release was therefore assessed. DPPE inhibited the release of arachidonic acid in response to collagen but had no effect on its subsequent conversion to thromboxane. The findings imply that intracellular histamine acts at an early stage in collagen-induced platelet activation, most likely prior to arachidonic acid release, and further that DPPE inhibits ADP and EP171-induced activation by a non-histamine related effect.