Formation of dissolved gaseous mercury (DGM) and its volatilization from aquatic systems can be considered as a natural attenuation process, which limits the methylation of mercury (Hg) and Hg accumulation in fish. Although gold-mining activity and erosion of tropical soils lead to increased Hg concentration in aquatic systems, little is known about DGM production and distribution in tropical aqueous systems. Our work explores the vertical distribution and principal sources of DGM in a meromictic Amazonian reservoir. Dissolved gaseous mercury measurements carried out in Petit-Saut reservoir (French Guiana, South America) revealed DGM increase in the surface waters and at the bottom layers of the reservoir during the dry season. As in arctic and temperate lakes, high DGM concentrations in surface waters were attributed to sunlight-mediated photochemical processes. Dissolved gaseous mercury concentrations in the anaerobic hypolimnion were larger than in temperate or arctic lakes. In order to elucidate Hg(II) reduction pathways in the bottom layer of tropical reservoir, laboratory Hg(II) reduction experiments were performed with anoxic aqueous suspensions of surface sediments either untreated or treated by gamma-ray and NaN3. Our results indicated that DGM production at the bottom layer of Petit-Saut reservoir was biologically mediated. Dissolved gaseous mercury formation rates in the surface sediment suspensions were of the same order of magnitude as formation rates in freshwater lakes reported in literature.