Pharmacological modulation of [K+]oaccumulation and action potential changes during acute myocardial ischemia is under evaluation as a promising new antiarrhythmic and cardioprotective strategy during myocardial ischemia and reperfusion. We studied the effects of cromakalim, a K+channel opener that activates ATP-sensitive K+channels, in isolated arterially perfused rabbit interventricular septa subjected to ischemia and reperfusion and, through use of the patch clamp technique, in inside-out membrane patches excised from guinea pig ventricular myocytes. During aerobic perfusion, 5 μM cromakalim shortened action potential duration (APD) from 217±7 to 201±10 msec, had no effect on [K+]o, and reduced tension by 17±3% (n=11). During ischemia, pretreatment with 5 μM cromakalim resulted in 1) more rapid APD shortening (71±9 versus 166±7 msec at 10 minutes and 63±12 versus 122±8 msec at 30 minutes), 2) similar [K+]oaccumulation after 10 minutes (8.9±0.3 versus 9.6±0.5 mM) but a trend toward increased [K+]oaccumulation after 30 minutes (11.0±1.7 versus 9.6±1.0 mM), and 3) similar times for tension to decline to 50o of control (2.14±0.16 versus 2.14±0.19 minutes) but shorter time to fall to 20% of control (4.34±0.33 versus 4.90±0.22 minutes;p=0.003). After 60 minutes of reperfusion following 30 minutes of ischemia, recovery of function was similar, with a trend toward better recovery of developed tension (to 58±9%o versus 39±10% of control; p=0.18) and tissue ATP levels in cromakalim-treated hearts but no differences in APD or rest tension. Thus, 5 μM cromakalim had mild effects in normal heart but greatly accelerated APD shortening during ischemia without markedly increasing [K+] accumulation, possibly because the more rapid APD shortening reduced the time-averaged driving force for K+efflux through ATP-sensitive K+channels. A significant cardioprotective effect during 30 minutes of ischemia plus 60 minutes of reperfusion could not be demonstrated in this model. In excised membrane patches studied at room temperature, the ability of cromakalim to activate ATP-sensitive K+channels was significantly potentiated by 100 μM but not 15 μM cytosolic ADP, suggesting that in addition to the modest fall in cytosolic ATP during early ischemia, the rapid increases in cytosolic ADP may further sensitize cardiac ATP-sensitive K+channels to activation by cromakalim. This factor may contribute to the marked acceleration of APD shortening during ischemia produced by concentrations of cromakalim that have minimal effects on APD in aerobic perfused tissue. Activation of ATP-sensitive K+channels by cromakalim was not significantly affected by other ischemic factors such as lactate (20 mM), Pi(10 mM), or acidosis (pH 6.5).