Using Mg2+ macroelectrodes based on the sensor ETH 7025 and accurate Mg2+-EDTA buffer solutions, the apparent Mg2+-ATP dissociation constant (Kapp) was measured at 25 and 37 degrees C in background solutions mimicking the cationic intracellular milieu of muscle cells. The mean +/- SD (in microM) at 25 degrees C was 157.0 +/- 13 (n = 4), 127.5 +/- 12.0 (n = 11), 101.0 +/- 9.0 (n = 4) and at 37 degrees C was 106.6 +/- 9.6 (n = 4), 87.4 +/- 4.9 (n = 4), 78.1 +/- 2.0 (n = 4) at pH values of 6.7, 7.2, and 7.7, respectively. The dependence of Kapp at 25 degrees C on the ionic strength was also measured, the mean +/- SD (microM) being 61.9 +/- 2.2 (n = 3), 127.5 +/- 12 (n = 11), and 243.0 +/- 11.8 (n = 3) at ionic strengths of 0. 087, 0.156 (normal background), and 0.3 m, respectively. These values are larger than the Kapp values most commonly used in the literature (87.4 microM compared to 38 microM at pH 7.2 and 37 degrees C) to estimate the [Mg2+]i in 31P NMR experiments, attributed to the difficulties in setting the [Mg2+]i without the use of Mg2+ buffer solutions. If these new values are used, the literature values for [Mg2+]i estimated by 31P NMR increase by a factor of around 1.5, making them similar to values obtained by direct Mg2+ microelectrode measurements.