A novel ion selective electrode (ISE) for ionized magnesium (IMg2+) in whole blood (WB), plasma (PL) and serum (S) has now been designed and characterized in normal human subjects, diseased and pregnant subjects. Using this ISE on various levels of aqueous Mg2+ solutions (0.1-3.0mM), mean Mg2+ values are within 94.6 and 99.2% of their targets. The linearity of the ISE (0.1-3.0mM) in aqueous solution and human PL and S ranges between 92.0 and 99.3%. The ISE is highly selective for IMg2+, yielding measurements in less than 2 min, and exhibiting no or negligible effects from physiologic concentrations of Ca2+,Na+,K+ or H+. Ligand binding studies indicate that pathophysiologic concentrations of most smaller molecular weight anions fail to interfere with IMg2+ measurements in aqueous solution, PL or S. Likewise, pathophysiologic concentrations of heavy metals or lipids do not interfere with measurements for IMg2+. Comparison of healthy, normal WB, PL and S IMg2+, using the ISE, with measurements of ultrafilterable Mg by atomic absorption spectroscopy (AAS) are excellent. The mean value for IMg2+ in normal WB, PL and S is 0.58-0.60mM (range = 0.53-0.67mM). Compared to total Mg (TMg) (0.75-0.96mM) and ICa2+ (1.10-1.30mM), IMg2+ is held in a narrow, tight range, representing 71% of TMg. Preliminary studies on plasma and serum IMg2+ during open heart surgery and term pregnancy demonstrate significant abnormalities in IMg2+ and % IMg2+. The new ISE for IMg2+ should prove invaluable and revolutionary in studies of Mg metabolism in both healthy and diseased states.