Objective Effects of melatonin and serotonin on ethanol induced ulceration in the rat stomach were investigated. Methods Serotonin was injected subcutaneously, then melatonin solution and finally ethanol was put into the ex vivo gastric lumen to prepare the experimental models. Glandular mucosal blood flow (GMBF) and the gastric mucosal injury were observed. Results Melatonin and serotonin administration did not induce observable gastric mucosal of damage in the ex vivo stomach, but the serotonin reduced glandular mucosal blood flow (GMBF) with dose dependence in this organ. Ethanol reduced GMBF and induced visible glandular mucosal damage. The latter effect was prevented by melatonin pretreatment. Serotonin pretreatment aggravated the gastric mucosal injury and GMBF changes induced by ethanol, but these effects were partially reversed by melatonin. Conclusion The findings indicate that the GMBF and gastric injury are related, the reduction in GMBF, however, may not be the sole factor responsible for ulceration. The antagonistic effects of melatonin on serotonin action on the stomach suggest that melatonin may play a modulator role for serotonin action on the gastrointestinal tract.