Stimulation of human platelets with endothelin raises cytosolic pH (pH i). In order to determine whether this effect is mediated via protein kinase C and Na +-H + linked pathways, the effects of staurosporine and calphostin C (protein kinase C inhibitors) and 5-(N,N-hexamethylene) amiloride (Na +-H + exchange blocker) on endothelin-induced pH i responses in human platelets were assessed. In addition, platelet endothelin receptor subtypes were determined pharmacologically using the selective ET A receptor antagonist BQ-123 and the ET B receptor agonist sarafotoxin S6c. pH i was measured spectrofluorometrically using the fluorescent probe BCECF-AM in platelets obtained from 15 healthy subjects. Endothelin-1 at a fixed concentration of 10 −9 M significantly increased pH i from 7.11 ± 0.01 ([H +] i = 77 ± 0.9 nM) to 7.19 ± 0.04 ([H +] i = 64 ± 0.9 nM) (p<0.01). The pH i-stimulating effect of endothelin-1 was inhibited by 10 −7 M staurosporine, calphostin C and 5-(N,N-hexamethylene) amiloride. BQ-123 (10 −7 M) abolished the pH i responses to endothelin-1, whereas sarafotoxin S6c had no effect on platelet pH i. These data suggest that in vitro effects of endothelin-1 on platelet pH i are receptor-mediated via a pathway involving protein kinase C. Platelet endothelin receptors appear to be of the ET A subtype.