We recently demonstrated the presence of melatonin in ram seminal plasma and differences in its concentration in this fluid between the breeding and nonbreeding season. In this study, we investigate the hypothesis that in vitro treatment with melatonin affects ram sperm quality, and that this is reflected in the in vitro fertilization (IVF) results. Semen from nine rams was collected during the nonreproductive season and treated with 1 mum, 10 nm and 100 pm melatonin. Samples were incubated at 39 degrees C and 5% CO2, and motility, viability, capacitation status and phosphatidylserine (PS) translocation were assessed before and after melatonin addition, either 1 or 3 hr of incubation. Fertility rate of the melatonin-treated samples was determined by means of IVF. Although melatonin failed to affect both sperm kinematic parameters and viability, the exposure of ram spermatozoa to melatonin has a direct effect, decreasing capacitation and PS translocation at 1 mum, and increasing short-term capacitation at 100 pm, which caused an increased oocyte fertilization rate following IVF. Furthermore, cleavage rate of oocytes fertilized with 100 pm melatonin-treated spermatozoa was higher than that with 1 mum melatonin and control samples (P < 0.1). These results prove that melatonin has a direct effect on ram spermatozoa in the nonreproductive season, which can be explained, at least in part, by the melatonin capacity as a reactive oxygen species scavenger and antioxidant. These findings might help to select the optimal experimental conditions for IVF and to improve sperm preservation protocols.