The effect of extracellular Na+ [( Na+]e) removal on agonist-induced granule secretion in platelets in relation to [pH]i and [Ca2+]i changes was investigated. Substitution of [Na+]e with choline+ of K+ resulted in a significant enhancement of 5HT secretion induced by thrombin, collagen, U46619 and the protein kinase C activators, PMA and diC8. Increases in [Ca2+]i induced by thrombin and U46619 were slightly inhibited or unaffected in these buffers, but [pH]i increases induced by thrombin, U46619, PMA and diC8 were abolished and a drop in [pH]i (0.05 0.1 units below resting) was observed. Although preincubation with potassium acetate produced a big drop in [pH]i and greatly increased secretion with all the agonists, particularly in the absence of [Na+]e, clear evidence that [pH]i rises due to Na+/H+ exchange are inhibitory to secretion was obtained only with thrombin. Thus, (i) NH4Cl, which restored the increase in [pH]i in the absence of [Na+]e reduced the potentiated secretory response to thrombin, (ii) no increase in thrombin-induced secretion was observed when Na+ was replaced with Li+, which allowed a normal increase in [pH]i and (iii) ethyl isopropyl amiloride (EIPA) abolished the [pH]i rise and potentiated thrombin-induced secretion. With collagen and U46619, the results suggest that removal of [Na+]e per se rather than inhibition of Na+/H+ exchange results in enhanced secretion. It is concluded that [Na+]e per se and [pH]i elevations via Na+/H+ exchange both have important inhibitory roles in the control of platelet granule secretion.