C-terminal fragments of the sulphonylurea receptor SUR2A can alter the functional expression of cloned ATP- sensitive K⁺ channels (K_ATP). To investigate the protective role of K_ATP channels during metabolic stress we transfected SUR2A fragments into adult rat cardiac myocytes. A fragment comprising residues 1294-1358, the A-fragment, reduced sarcolemmal K_ATP currents by over 85% after two days (pinacidil-activated current densities were: vector alone 6.30 ± 0.85; A-fragment 0.94 & [plusmn]0.07 pA pF^-1, n=6,6, p<0.001). An inactive fragment (1358-1545, current density 7.04 & [plusmn] 1.22 pA pF^-1, n=6) was used as a control. During metabolic inhibition (CN and iodoacetate) of isolated myocytes stimulated at 1 Hz, the A-fragment delayed action potential shortening and contractile failure, but accelerated rigor contraction and increased Ca²⁺ loading. On reperfusion, A- fragment transfected cells also showed increased intracellular Ca²⁺ and the proportion of cells recovering contractile function was reduced from 40.0% to 9.5% (p<0.01). The protective effect of pretreatment with 2,4-dinitrophenol, measured from increased functional recovery and reduced Ca²⁺ loading, was abolished by the A- fragment. Our data are consistent with a role for K_ATP channels in causing action potential failure and reduced Ca²⁺ loading during metabolic stress, and with a major role in protection by preconditioning. The effects of the A-fragment may arise entirely from reduced expression of the sarcolemmal K_ATP channel, but we also discuss the possibility of mitochondrial effects.