To investigate various factors that possibly affect oxygen affinity of hemoglobin (Hb-O2 affinity) in chronic hemodialysis (HD) patients, we determined P50 at standard condition (P50std), 2,3-diphosphoglycerate (2,3-DPG) content in red blood cells, serum inorganic phosphorus (S-Pi), Hb, and arterial blood gas analysis in 55 HD patients. P50std in HD patients was higher than that in normal controls (26.6 ± 1.6 vs. 25.4 ± 1.4 mm Hg; p < 0.001). We could find neither an effect of alkalizating agents for HD (acetate vs. bicarbonate) nor an effect of the underlying disease (diabetics vs. nondiabetics) on Hb-O2 affinity. There were significant positive correlations between P50std and the duration of HD therapy (r = 0.598; p < 0.001) and between P50std and SPi (r = 0.476; p < 0.001), contrasting with the absence of correlation between P50std and Hb. Forward stepwise multiple-regression analysis demonstrated that the duration of HD therapy played the most important roles in determining P50std, followed by SPi and PO2. These data suggest that the major factor influencing Hb-O2 affinity in chronic HD therapy is the duration of the therapy and that the minor factors are SPi and PO2.