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High performance liquid chromatography with electrochemical detection was developed for the determination of serotonin (5-hydroxytryptamine) in rabbit platelet and plasma. A peak from tissue extract (peak a) was identified as serotonin based on retention time on the chromatogram, using a potential that gave limiting current on both a current-potential curve and gas chromatography-mass spectrum, as compared with that of authentic serotonin. For the assay of serotonin, 5-hydroxy-N-methyltryptamine was used as an internal standard and the extraction recovery of the serotonin added to the platelet-rich plasma was almost total. Furthermore, using this highly sensitive method, it has been demonstrated that the release of endogenous serotonin from rabbit platelets occurs during collagen-induced platelet aggregation but not during adenosine diphosphate-induced platelet aggregation. The procedures described should prove to be simple and useful for pharmacological and physiological investigations on the relationship between platelet aggregation and release reaction.