Current literature suggests that the platelet 5-HT2 receptor, thought to be the only active platelet serotonin (5-HT) receptor, may be both heterogeneous and not the sole 5-HT receptor subtype on platelet membranes. The present studies used more selective tryptamine agonists, and the antagonists ketanserin and SCH 23390 to characterize rat platelet 5-HT receptors in vitro. The present studies also addressed anticoagulant effects (citrate versus heparin) on platelet 5-HT aggregation in the rat. 5-HT was less potent at enhancing ADP-induced aggregation in heparinized rat platelet rich plasma (PRP) as compared to citrated PRP. However, potency and maximum aggregation to ADP were greater in heparinized platelets. In citrated rat PRP, the selective tryptamine agonists, 5-carboxamidotryptamine (5-CT) and 2-CH3-5-HT, produced little change in the baseline ADP-induced aggregation and induced platelet shape change only in higher concentrations (greater than 1 microM). In contrast, alpha-CH3-5-HT-induced shape change and enhancement of ADP aggregation were superimposable with that of 5-HT itself suggesting 5-HT2 receptor activation. The antagonists, ketanserin and SCH 23390, inhibited 5-HT enhancement of ADP-induced aggregation with affinity constants consistent with the presence of 5-HT2 receptors as well. Studies with heparinized rat PRP did not unmask activity to 5-CT or 2-CH3-5-HT. Thus, although reports of multiple platelet 5-HT receptors exist, the only detectable, functional 5-HT receptor to enhance aggregation in rat platelets was probably of the 5-HT2 type.