We have examined the effect of exposing isolated rat ventricular myocytes to lithium while measuring cytosolic free magnesium ([Mg2+]i) and calcium ([Ca2+]i) levels with the fluorescent, ion sensitive probes mag-fura-2 and fura-2. There was a significant rise in [Mg2+]i after a 5 min exposure to a solution in which 50% of the sodium had been replaced by Li+, but not when the sodium had been replaced by bis-dimethylammonium (BDA). However, there were significant increases in [Ca2+]i when either Na+ substitute was used. The possibility that Li+, which enters the cells, interferes with the signal from mag-fura-2 was eliminated as Li+ concentrations up to 10 mM had no effect on the dye's fluorescence signal. A possible explanation for these findings is that Li+ displaces Mg2+ from intracellular binding sites. Having considered the binding constants for Mg2+ and Li+ to ATP, we conclude that Li+ can displace Mg2+ from Mg-ATP, thus causing a rise in [Mg2+]i. This work has implications for other studies where Li+ is used as a Na+ substitute.