The free intracellular Mg2+ concentration ([Mg2+]i) was investigated in frog sartorius and crayfish phasic and tonic skeletal muscle fibres, using a new Mg2(+)-sensitive microelectrode based on the ionophore ETH 5214 [Hu et al. (1989) Anal Chem 61:574-576]. In Ringer solution containing 0.5 mmol/l MgCl2, the mean [Mg2+]i of the frog muscle fibres was 1.3 mmol/l. In phasic crayfish muscle fibres, [Mg2+]i was about twice as high (mean 3.5 mmol/l) as in tonic fibres (mean 1.5 mmol/l), measured in van Harreveld solution containing 1.2 mmol/l MgCl2. Long-lasting (3-12 h) incubation of frog skeletal muscle fibres in Na(+)-free solution produced a reversible increase of [Mg2+]i by a factor of about 1.7. A tenfold rise of extracellular Mg2+ led to an increase in [Mg2+]i in the presence as well as in the absence of Na+. In these experiments, mean [Mg2+]i values of 3.2 mmol/l were never exceeded. Thus, [Mg2+]i remained at least 60 times lower than predicted from a passive distribution across the cell membrane. The results suggest the existence of a Na(+)-dependent and a Na(+)-independent Mg2+ extrusion mechanism, which is regulated by actual Mg2+ concentrations.