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The effects on serum ionized calcium (ICa) and pH of different storage conditions of blood or serum, and of physiological influences such as over-ventilation, food intake and dietary sodium intake have been investigated. Temperature and time-related changes in ICa occurred with storage and were minimized by immediate separation of serum and storage at 4°C, (6 h or less). Elevation of serum ICa and a fall in pH accompanied increased salt intake; over-ventilation induced an elevation of serum pH and a reduction in ICa. Day-to-day intra-individual variation of ICa was 0.93%.We proceeded to examine a group of age-, sex- and race-matched hypertensive and normotensive subjects under standardized conditions designed to minimize such technical and physiological artefacts. ICa was not significantly different in the two groups; however, serum pH was significantly elevated in the hypertensive group. In the combined group of normotensive and hypertensive subjects, serum pH was significantly correlated with blood pressure. Exclusion of the black subjects from the analysis did not alter the findings.