Introduction: We have previously reported that TRPV1 activation is required for a secondary plateau phase of adenosine diphosphate (ADP)-evoked Ca2+ release in human platelets, through its ability to control serotonin release from these cells1. Here we have further examined the link between TRPV1 activation and serotonin secretion in ADP-stimulated platelets. Methods: Platelets were isolated from blood obtained by venepuncture of healthy volunteers under informed consent and with local ethical committee approval in accordance with the Declaration of Helsinki. Changes in cytosolic pH (pHcyt), cytosolic Ca2+ concentration ([Ca2+]cyt), intracellular Ca2+ store concentration ([Ca2+]st) and acidic organelle pH (pHao) were measured in BCECF-, Fura-2-, Fluo-5N- and lysosensor green-loaded human platelets respectively. Platelets were stimulated with either 100 µM capsaicin or 50 µM ADP in the absence of extracellular Ca2+ at 37°C. Data are presented as mean ± S.E.M. of the number of samples (n) indicated. Statistical significance was tested by Student’s t-test. Results: Stimulation of platelets with capsaicin in the absence of extracellular Ca2+ elicited a small rise in [Ca2+]cyt, as we have previously reported1. Under the same conditions capsaicin also elicited an acidification of the cytosol, an alkalinisation of the acidic organelles and a small decrease in [Ca2+]st (n=3). Stimulation of platelets with ADP elicited the same effects, which could be partially prevented by treatment of platelets with the TRPV1 inhibitor, 5′-Iodo-resiniferatoxin (20 µM, n= 7-14; p<0.05). Conversely, concurrent stimulation of platelets with ADP and capsaicin potentiated the changes in [Ca2+]cyt (131.2 ± 8.7 % of control; n = 6; p < 0.05), [Ca2+]st (175.0 ± 19.1% of control; n = 6; p < 0.05), pHao (296.4 ± 75.3% of control; n = 6; p < 0.05) and pHcyt (291.3 ± 52.5 % of control; n = 6; p < 0.05). Previously we have demonstrated that pretreatment of platelets with ketanserin (a 5-HT2A receptor antagonist that inhibits autocrine serotonin signalling) significantly inhibited ADP-evoked rises in [Ca2+]cyt1. We therefore investigated whether this might be due to an effect on the ADP-evoked alkalinsation of the acidic organelles. Pretreatment for 2 minutes at 37°C with 25 µM ketanserin inhibited the ADP-evoked changes in [Ca2+]st (31.1 ± 6.2% of control; n = 5; p < 0.05), pHao (56.9 ± 14.3% of control; n = 7; p < 0.05) and pHcyt (59.4 ± 7.3% of control; n = 6; p < 0.05). Conclusions: These data suggest that TRPV1 regulates ADP-evoked serotonin secretion from human platelets. Autocrine serotonin signalling then elicits Ca2+ release from the acidic organelles, potentiating the initial ADP-evoked Ca2+ signal.