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Background and objectives Circulating levels of anions that bind to magnesium and calcium are often altered in patients with CKD. However, it is unknown how these alterations affect the ionized fraction of magnesium and calcium. Design, setting, participants, & measurements This cross-sectional study involved patients on maintenance hemodialysis and patients not on dialysis who visited the outpatient department of nephrology. We collected whole-blood samples to measure ionized magnesium and calcium concentrations. Adjusted anion gap was calculated as an integrative index of unmeasured anions. Results A total of 118 patients on hemodialysis and 112 patients not on dialysis were included. Although the prevalence of hypermagnesemia defined by total magnesium was much higher in patients on hemodialysis than in patients not on dialysis (69% versus 12%; P Conclusions Anions that accumulate in patients on hemodialysis contribute to the lower ionized fraction of magnesium and calcium. Equations that incorporate the anion gap provide better predictions of ionized magnesium and calcium in patients on hemodialysis.