The recovery (%) of the left ventricular developed pressure by (S)-(−)-pyrapyridolol (5×10 −8 M) (90.7%), an optical isomer of a new 5-HT 1A receptor antagonist, was greater than that by (R)-(+)-pyrapyridolol (66.2%, control: 34.4%) against ischemia–reperfusion injury in perfused Langendorff guinea-pig hearts. In the perfused mitochondrial preparation, (S)-(−)-pyrapyridolol inhibited the mitochondrial Ca 2+ (Cam) elevation that was brought about by the change of Ca 2+ content or pH of perfusate, similar to findings with cyclosporin A, well known to be an inhibitor of the mitochondrial permeability transition pore (MPTP). The mitochondrial K ATP channel opener, diazoxide, also inhibited the Cam elevation, but the mitochondrial K ATP channel antagonist, 5-hydroxydecanoic acid, attenuated it. There were significantly fewer numbers of TUNEL-positive cells in these (S)-(−)-pyrapyridolol-treated hearts than the control or (R)-(+)-pyrapyridolol, with decreases of the caspase-3 activity. Therefore, these results suggest that (S)-(−)-pyrapyridolol likely inhibits the opening of the MPTP by preventing the Cam overload induced apoptosis related to endogenous 5-HT accumulation in ischemia–reperfusion hearts.