Using rat ventricular cells, we studied the actions of free fatty acids and their ability to modulate the ATP-sensitive K+ channel and to activate a new type of ATP-insensitive K+ channel previously identified in rat atrial cells. Perfusion of the cytoplasmic face of the membrane with unsaturated fatty acids (10-50 microM) such as arachidonic, linoleic, and eicosatrienoic acids inhibited the ATP-sensitive K+ channel almost completely; lysophospholipids also markedly inhibited this channel. Inhibition was due to decreases in the frequency and the burst duration of channel openings. Arachidonic acid activated the ATP-insensitive K+ channel with an outwardly rectifying property. Since the level of free fatty acids rises after longer periods of ischemia, we speculate that the ATP-insensitive K+ channel contributes to the late or secondary phase of extracellular K+ accumulation.