K+ currents were recorded from ATP-sensitive channels in inside-out patches from isolated rat ventricular myocytes. In the absence of internal divalent cations the current voltage relationship could be described by constant-field assumptions with a permeability of 1.25 X 10(-13) cm2/s; outward currents saturated under a high driving force for K+ movement. Internal 0.1-5.0 mM Mg2+, 0.1 microM Ca2+ and 10 mM Na+ each depressed the flux of K+ ions moving outwards through open channels. Internal 0.1-5.0 mM Mg2+, 0.1-1.0 microM Ca2+ and 1-10 microM Ba2+ and Sr2+ blocked K+ channel activity in a dose- and voltage-dependent manner. Run-down channels could be reactivated by Mg-ATP, but not by AMP-PNP, ATP gamma S or Mg-free ATP which suggested that phosphorylation of the channels was involved in their activity. Ca2+ (greater than = 1 microM) and Sr2+ (1 mM) markedly inactivated K+ ATP channels, millimolar Ba2+ or Mg2+ were less effective. This suggested that the run down of the channels was a Ca2+-dependent dephosphorylation of the K+ channel protein.