We used the hamster cheek pouch microcirculation to investigate the effects of melatonin (ME) on ischemia reperfusion (I-R) injury by in vivo microscopy. ME is a hormone produced by the pineal gland and is the most powerful and effective hydroxyl radical scavenger detected to date in vitro. The second aim was to determine the scavenger effect of ME in cheek pouch microcirculation when topically applying an oxygen-derived free radical generating system. Ischemia was induced by clamping the cheek pouch for 30 min followed by 30 min of reperfusion. We quantified the increase in permeability, the perfused capillary length and leukocyte adhesion by computerized methods. Microcirculation was also exposed to a hypoxanthine-xanthine oxidase (H-X) system. In control hamsters I-R was associated with increased permeability, increased number of leukocytes sticking to venules, and decreased perfused capillary length. Treatment with ME completely inhibited microvascular edema formation and reduced the number of leukocytes sticking to venules after reperfusion. Moreover, ME prevented the marked decrease in perfused capillary length, preserving microvascular perfusion. ME topically applied reduced significantly the permeability increase due to H-X exposure. The beneficial effect of ME may be related to its antioxidant properties. These protect the endothelial barrier integrity as well as preserve microvascular blood perfusion by dysfunctions after I-R.