BACKGROUND Activation of either protein kinase C or the ATP-sensitive potassium channel has been shown to induce the preconditioning response. RESULTS To investigate whether preconditioning activation adjuvant to hypothermic blood cardioplegia enhances postischemic contractile recovery, 23 adult (0.5 to 1.0 year old) sheep were randomized to receive cardioplegia based on the hyperpolarizing ATP-sensitive potassium channel opener pinacidil, the protein kinase C activator 4 beta-phorbol-12, 13-dibutyrate (PDBu), or standard potassium-magnesium (K1/Mg2+). All groups underwent 60 minutes of 10 degrees C antegrade intermittent blood cardioplegia and 30 minutes of reperfusion. Mechanics were assessed on modified right heart bypass by the preload recruitable stroke work relation and the end-systolic pressure-volume relation. Diastolic function was modeled by the exponential time constant of isovolumic left ventricular pressure decay (Tau) and the "stiffness" coefficient (beta) of the end-diastolic pressure volume relation. Recovery rhythm was defined by the first electrical activity seen during reperfusion. Cardioplegic arrest and preconditioning induced by pinacidil provided superior recovery of contractile function compared with PDBu (100% versus 57% recovery, P < .05) or K+/Mg+ (100% versus 56% recovery, P < .05). Active (Tau) and passive (beta) diastolic function was preserved by all three arrest modalities. Hearts treated with pinacidil demonstrated a rapid recovery of a coordinated contraction pattern, which was offset by a reperfusion tachycardia, whereas PDBu arrest was associated with ventricular fibrillation on reperfusion. CONCLUSIONS Preconditioning during cardioplegic arrest is agent specific, feasible at cold temperatures, and may be superior to the use of standard K+/Mg2+ cardioplegia.