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One of the major physiological effects of melatonin is coupling the internal clock with different organ functions. Despite the long list of functional responses to melatonin discovered in the past, it is not clear when responsiveness to melatonin develops during ontogeny. Thus, the aim of the present study was to investigate when Muscovy duck and chicken embryos begin to exhibit heart rate (HR) changes to exogenous melatonin. HR was recorded continuously in Muscovy duck embryos from day 24 of incubation (D24) and in chicken embryos from D17 until hatching. Every day four doses of 10 µg melatonin were injected into each egg at 30-min intervals. In Muscovy duck embryos, HR responses to melatonin were first observed on D25; from D27 all embryos responded. In all cases, HR decreased immediately after the injection. HR deviation from baseline values and duration of decreased HR period increased during the experimental period. Chicken embryos showed similar responses as Muscovy ducks from D17 onwards; from D18 the response rate was 100%. On D19, dose-response data revealed a partial responsiveness to exogenous melatonin at doses of 0.1 to 1 ng and full responsiveness from 10 ng to 10 µg. The time of the first occurrence of HR responses to melatonin coincides with published results on the start of periodic pineal melatonin secretion. These data suggest that the output signal of the avian internal clock, periodic plasma melatonin fluctuations, could already prenatally result in periodic cardiac function in these two avian species.