Caffeine has been extensively used to study intracellular Ca2+ control and contraction-relaxation in cardiomyocytes. The effects of caffeine on intracellular free Mg2+ concentration, [Mg2+]i, were studied in isolated adult rat ventricular myocytes by fluorescent techniques using mag-fura-2. Basal [Mg2+]i was 0.62 +/- 0.02 mM, n = 54, in quiescent cells and 0.73 +/- 0.02 mM, n = 23, in electrically-stimulated adult rat ventricular myocytes. Caffeine, 20 mM, significantly decreased [Mg2+] in both quiescent (-0.17 +/- 0.01 mM) and electrically-stimulated (-0.16 +/- 0.01 mM) adult ventricular myocytes. Ryanodine, a blocker for Ca(2+)-release channels of the sarcoplasmic reticulum, did not have any effect on basal [Mg2+]i, 0.67 +/- 0.02 mM nor on caffeine-induced reduction in [Mg2+]i, -0.16 +/- 0.01 mM in quiescent cardiomyocytes or electrically-stimulated cells; 0.74 +/- 0.03 mM and -0.11 +/- 0.03 mM, respectively. Ruthenium red, an inhibitor of mitochondrial Ca2+ uptake, also failed to alter basal [Mg2+]i, or caffeine-induced reduction in [Mg2+], in either quiescent or electrically-stimulated cells. The effects of caffeine on [Mg2+]i, may be important in considering the use of this drug to study contraction/function studies in heart cells.