Cryopreservation damages permeability of sperm mitochondrial membranes, with formation of a mitochondrial permeability transition pore (mPTP). Mitochondria are both a primary synthesis site and principle target for melatonin, which can directly inhibit mPTP formation. The objective was to determine effects of melatonin on mPTP opening of frozen-thawed ram sperm and elucidate underlying pathways by antagonist and agonists of melatonin receptors (MTs), and antagonists of PI3K and GSK 3 beta treatments; furthermore, plasma membrane integrity, mitochondrial membrane potential (Delta psi m), mitochondrial cytochrome c (Cyt c) release and fertilization were analysed to assess the effect of mPTP status mediated by melatonin on quality of frozen-thawed sperm. Fresh ram semen was diluted in glucose-egg yolk buffer with 0 or 10(-7) M melatonin (frozen and frozen + melatonin groups, respectively) and slow-frozen. In frozen-thawed sperm, melatonin added at initiation of 4 degrees C equilibration was most effective for inhibiting mPTP opening, decreasing peptidyl-prolyl-cis/trans isomerase activity of cyclophilin D and increasing plasma membrane integrity, Delta psi m, mitochondrial Cyt c concentration and fertilizing ability (p .05). In a mechanistic study, the melatonin receptor (MT)1 antagonist eliminated inhibition of melatonin on mPTP opening, whereas MT1 agonist had opposite effects (p < .05). Neither MT2 antagonist nor agonist had significant effect, but PI3K and/or GSK 3 beta antagonist decreased inhibition of MT1 agonist on mPTP opening (p .05). In conclusion, melatonin improved sperm cryopreservation, perhaps by acting on MT1 via the PI3K-Akt-GSK 3 beta pathway to inhibit mPTP opening.