Elementary Na + currents were recorded in inside-out patches excised from cultured neonatal rat heart myocytes in order to study the influence of cytosolic Mg + + and other bivalent cations present at the cytoplasmic membrane surface on cardiac Na + channel gating. Exposing the cytoplasmic membrane surface to a Mg + +free environment shortened the open state of cardiac Na + channels significantly. Zop,, declined to 62_+2% of the value obtained at 5 mmol/1 Mg + +. Other channel properties including the tendency to reopen and the elementary current size either changed insignificantly within a 10% range or remained completely unchanged. An almost identical change of Zop~, can be caused by switching from a Mn ++ (5 mmol/1) containing internal solution to a Mn++-free internal solution. But Zopo, failed to significantly respond to a variation in internal Ni ++ from 5 mmol/1 to 0 mmol/1. The same response to internal Mg + + withdrawal was obtained with (-)-DPI-modified, non-inactivating Na + channels, indicating that the exit rate from the open state remains as sensitive to cytosolic Mg + + variations as in normal Na + channels with operating inactivation.