In this study we quantified greenhouse gases concentration (nitrous oxide and methane) and other physicochemical parameters in the water column of Prado eutrophic reservoir to determine with in situ experiments the nitrous oxide source and the vertical distribution of both gases. The results indicated that nitrification contributed more actively than denitrification to nitrous oxide production in the water column to estimated rates of 122.5 ± 79.9 nM/d, and that, in comparison with other dams and lakes in the world, the nitrous oxide (up to 38.6 ± 0.9 nM) and methane concentrations (up to 242 ± 85.5 µM) measured in the reservoir were high. This is the first study in the Prado dam showing the oversaturation of both gases in the water column, which suggests that this reservoir could be emitting significant quantities of greenhouse gases to the atmosphere. © Acad. Colomb. Cienc. Ex. Fis. Nat. 2015.