1. The effect of prolonged application of isoprenaline on intracellular free-Mg2+ concentration ([Mg2+]i) was examined by use of 31P-nuclear magnetic resonance (31P-n.m.r.) in rat isolated hearts. Left ventricular pressure (LVP) was simultaneously measured. 2. [Mg2+]i was estimated from the separation of the alpha- and beta-ATP peaks, using the dissociation constant of MgATP 38 microM (established previously). In normal (phosphate-free, Krebs-Henseleit) solution, [Mg2+]i was approximately 0.4 mM. 3. When isoprenaline was applied for 100 min, a transient increase in [Mg2+]i was observed during the initial 25 min, whilst concentrations of ATP ([ATP]) and phosphocreatine ([PCr]) decreased and [Pi] correspondingly increased. During the subsequent 75 min of isoprenaline application, [Mg2+]i decreased below its resting levels. Washout of isoprenaline restored [Mg2+]i and [PCr], but [ATP] remained low. These changes elicited by isoprenaline were not observed in the presence of propranolol, a typical alpha-adrenoceptor blocker. 4. Isoprenaline increased both LVP and heart rate. The increased LVP and heart rate slowly returned to lower values during prolonged application of isoprenaline, but remained higher than those before application. 5. The transient rise in [Mg2+]i elicited by isoprenaline could be attributed to the decrease in [ATP] resulting in a release of Mg2+. The subsequent decrease in [Mg2+]i during the prolonged applications suggests that beta-adrenoceptor stimulation itself facilitates Mg(2+)-extruding mechanism(s).