Background/Aim: The biocompatibility of hemodialysis membranes has a substantial impact on the mortality of patients with end-stage renal failure. In the present study, the effects of hemodialysis on the intracellular amount of diadenosine pentaphosphate (Ap<sub>5</sub>A), a hydrophilic, anionic substance with a low molecular weight, was investigated. Methods: The intracellular Ap<sub>5</sub>A concentrations were measured before and after hemodialysis using either polyacrylonitrile (AN69; n = 10) or polysulfone (n = 23) membranes. Ap<sub>5</sub>A was isolated from platelets using affinity chromatography and reversed-phase chromatography methods. Results: The Ap<sub>5</sub>A concentrations were quantified by ultraviolet absorption at 254 nm. The Ap<sub>5</sub>A concentrations were significantly higher in platelets from the patients with end-stage renal failure as compared with the 21 healthy control subjects (136 ± 50 vs. 9 ± 6 fg/platelet; mean ± SEM, p < 0.01). Before hemodialysis, the intracellular Ap<sub>5</sub>A concentrations in platelets from 10 patients with end-stage renal failure using an AN69 membrane were not significantly different from those in platelets from 23 patients using a polysulfone membrane (93 ± 39 vs. 155 ± 70 fg/platelet). However, after a hemodialysis session, the intracellular Ap<sub>5</sub>A concentrations in platelets from patients with end-stage renal failure using an AN69 membrane were significantly lower as compared with those in platelets before hemodialysis (51 ± 18 vs. 93 ± 39 fg/platelet, p < 0.05) as well as compared with those in platelets from patients using a polysulfone membrane (51 ± 18 vs. 250 ± 59 fg/platelet, p < 0.05). Conclusions: It was found that hemofiltration by using an AN69 membrane has a direct effect on the intracellular amount of Ap<sub>5</sub>A and that changes of intracellular hydrophilic substances are dependent on the hemodialysis membrane used.