Intracellular free magnesium ([Mg2+]i) was measured in isolated ferret papillary muscles using ion-selective microelectrodes filled with the new magnesium sensor ETH 5214. This new sensor, unlike its predecessor ETH 1117, does not react to marked changes in K+, Na+ or pH. Reducing Ca2+ from 20 microM to around 10 nM also did not affect the response so these electrodes are ideally suited to study intracellular Mg2+ and its regulation. The mean value for the [Mg2+]i from thirty-two experiments (forty-two impalements) was 0.85 mM, confirming previous estimates from this laboratory. Intracellular Mg2+ is not passively distributed and the possibility that Mg2+ is transported out of the cell by a Na(+)-Mg2+ exchanger was investigated. An increase in [Mg2+]o caused an increase in [Mg2+]i, as did stepwise reduction in the [Na+]o. However, this increase in [Mg2+]i on Na+ reduction also occurred in Mg2(+)-free solution suggesting that the increase in [Mg2+]i was due to the increase in intracellular Ca2+ on Na+ reduction. Moreover, increasing [Na+]i by strophanthidin did not change the [Mg2+]i and on increasing [Mg2+]o there was no reduction in the [Na+]i. Blocking ATP production lead to small increases in the [Mg2+]i. These results are not consistent with a Na(+)-Mg2+ exchanger as being the main outward transport mechanism for Mg2+ in this tissue.