The intracellular pH of platelets was measured by the fluorescent dye technique using 9-aminoacridine and BCECF-AM. Although platelet cytoplasmic pH determined by BCECF remained constant, the intracellular pH, determined by 9-aminoacridine, decreased during storage then increased when the stored platelets were incubated with fresh plasma, which led to the restoration of platelet functions. Since the average pH of cytoplasm and cell organelles is detected by 9-aminoacridine and only the cytoplasmic pH is detected by BCECF, the results suggested that changes of the organelle pH affected the platelet functions. Aggregability was measured using thrombin, ADP, collagen, A23187, arachidonic acid (AA), TPA and ristocetin, after the intracellular pH had been altered by treating the platelets with ionophores (monensin and nigericin) or after the membrane potential had been depolarized by treating with a buffer containing a high concentration of potassium ion. The depolarization increased platelet sensitivity only to three of the agonists, ADP, collagen and a low concentration of AA, while the increase of organelle pH and/or cytoplasmic pH enhanced the sensitivity to six of the above agonists, excluding ristocetin. Decrease of cytoplasmic pH reduced the sensitivity of the platelets to a low concentration of AA, but enhanced the sensitivity to thrombin and TPA. The results indicated that membrane potential, organelle pH and cytoplasmic pH influenced platelet functions via different mechanisms.