C-terminal fragments of the sulphonylurea receptor SUR2A can alter the functional expression of cloned ATP-sensitive K+ channels (KATP). To investigate the protective role of KATP channels during metabolic stress we transfected SUR2A fragments into adult rat cardiac myocytes. A fragment comprising residues 1294–1358, the A-fragment, reduced sarcolemmal KATP currents by over 85% after 2 days (pinacidil-activated current densities were: vector alone 7.04 ± 1.22; and A-fragment 0.94 ± 0.07 pA pF−1, n= 6,6, P < 0.001). An inactive fragment (1358–1545, current density 6.30 ± 0.85 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 KATP 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 KATP channel, but we also discuss the possibility of mitochondrial effects.