Background/Aims: The aim of this study is to measure the difference of ionized calcium between heparinized whole blood and serum. Methods: We recruited 107 maintenance hemodialysis (HD) patients from our hospital HD unit. The clinical and laboratory data included ionized calcium in serum and in whole blood (reference, 4.07 to 5.17 mg/dL). Results: The level of ionized calcium in serum was higher than that in whole blood (p < 0.001). Bland-Altman analysis showed that difference for ionized calcium was 0.5027. For the difference, the nonstandardized β was -0.4389 (p < 0.001)and the intercept was 2.2418 (p < 0.001). There was a significant difference in the distribution of categories of ionized calcium level between two methods (κ, 0.279;p < 0.001). Conclusions: This study demonstrates that whole blood ionized calcium is underestimated compared with serum ionized calcium. Positive difference increases as whole blood ionized calcium decreases. Therefore, significant hypocalcemia in whole blood ionized calcium should be verified by serum ionized calcium.