Recent in vivo experimental evidence suggests that isoflurane-induced cardioprotection may involve K-ATP channel activation during myocardial ischemia. The actual effect of isoflurane on cardioprotective ion conductance, however, such as that mediated by the opening of K-ATP channels, has been the subject of some controversy in the past. The investigation reported here used a patch-clamp technique to test the hypothesis that a metabolite of isoflurane, trifluoroacetic acid (TFA), contributes to isoflurane-induced cardioprotection via K-ATP channel activation. TFA enhanced channel activity in a concentration-dependent fashion, exhibiting half-maximal activation at 0.03 mM. TFA increased the number of openings of the channel, but did not affect the single channel conductance of K-ATP channels. Analysis of open and closed time distributions showed that TFA increased the burst duration and decreased the interburst interval without eliciting changes of less than 5 ms in open and closed time distributions. TFA diminished the ATP sensitivity of K-ATP channels in a concentration-response relationship for ATP. These results imply that TFA could mediate isoflurane-induced cardioprotection via KATP channel activation during myocardial ischemia and reperfusion.