Recent evidence demonstrates that long-chain acyl coenzyme A esters (CoAs) activate cardiac and beta-cell plasma-membrane (pmK(ATP)) adenosine triphosphate (ATP)-sensitive potassium channels. In this study, we have investigated the differential effects of acyl CoAs of short and medium side-chain length on cardiac and beta-cell pmK(ATP) isoforms. At the single-channel level, the addition of acyl CoAs of differing side-chain length (2 to 16 carbons) to the inside face of membrane patches from ventricular myocytes caused varying increases in pmK(ATP) channel open probability proportional to increases in acyl side-chain length (20 mumol/L acetyl CoA: 310% +/- 90%, 20 mumol/L decanoyl CoA: 570% +/- 150%). A similar dependence of activation on side-chain length was observed in recombinant pmK(ATP) channels (SUR2A/Kir6.2) with full activation of current requiring both the acyl and CoA moieties in the esterified form. We found the recombinant beta-cell K(ATP) channel (SUR1/Kir6.2) to be much less sensitive to medium-chain acyl CoAs (decanoyl CoA: 124% +/- 15% v 231% +/- 25% in SUR2A/Kir6.2), suggesting a role for the cardiac sulfonylurea receptor, SUR2A, in the molecular mechanism of activation by these compounds. We propose that fatty acid metabolism, and the resultant generation of acyl CoAs of varying side-chain length, may be an important regulator of cellular excitability via interactions with the K(ATP) channel.