Ion-sensitive microelectrodes (ISEs) have been used to measure intracellular [Mg2+] ([Mg2+]i) in cardiac muscle, although most measurements have tended to overestimate the value due to the poor selectivity of the Mg2+ ionophore in the sarcoplasm and to inaccurate collation of individual ISE measurements. This paper highlights the correct method for analysis of data from multiple ISE experiments. Since [Mg2+]i is constrained at a lower concentration than would be expected by passive distribution of the ion, some of the possible mechanisms underlying Mg2+ extrusion from ferret ventricular myocardium were investigated. During elevation of the extracellular [Mg], mean [Mg2+]i rose from 1.61 to 1.91 mM. The same intervention had no significant effect on membrane potential, intracellular [Na+] or pH measured with ISEs, and there was no change in resting [Ca2+], as assessed from fura-2 fluorescence. The data are not consistent with a simple mechanism for Na(+)-Mg2+ exchange as the primary mode of Mg2+ regulation in cardiac muscle or with an Mg2+ extrusion mechanism involving steady-state ion exchange.