Diazoxide and 5-hydroxydecanoate (5-HD; C10:0) are reputed to target specifically mitochondrial ATP-sensitive K+ (KATP) channels. Here we describe KATP channel-independent targets of diazoxide and 5-HD in the heart. Using submitochondrial particles isolated from pig heart, we found that diazoxide (10-100 μm) dose-dependently decreased succinate oxidation without affecting NADH oxidation. Pinacidil, a non-selective KATP channel opener, did not inhibit succinate oxidation. However, it selectively inhibited NADH oxidation. These direct inhibitory effects of diazoxide and pinacidil cannot be explained by activation of mitochondrial KATP channels. Furthermore, application of either diazoxide (100 μm) or pinacidil (100 μm) did not decrease mitochondrial membrane potential, assessed using TMRE (tetramethylrhodamine ethyl ester), in isolated guinea-pig ventricular myocytes. We also tested whether 5-HD, a medium-chain fatty acid derivative which blocks diazoxide-induced cardioprotection, was ‘activated’ via acyl-CoA synthetase (EC, an enzyme present both on the outer mitochondrial membrane and in the matrix. Using analytical HPLC and electrospray ionisation mass spectrometry, we showed that 5-HD-CoA (5-hydroxydecanoyl-CoA) is indeed synthesized from 5-HD and CoA via acyl-CoA synthetase. Thus, 5-HD-CoA may be the active form of 5-HD, serving as substrate for (or inhibiting) acyl-CoA dehydrogenase (β-oxidation) and/or exerting some other cellular action. In conclusion, we have identified KATP channel-independent targets of 5-HD, diazoxide and pinacidil. Our findings question the assumption that sensitivity to diazoxide and 5-HD implies involvement of mitochondrial KATP channels. We propose that pharmacological preconditioning may be reelated to partial inhibition of respiratory chain complexes.