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<p>Nitrous oxide (N₂O) is a greenhouse gas and an ozone depletion agent. Estuaries that are subject to seasonal anoxia are generally regarded as N₂O sources. However, insufficient understanding of the environmental controls on N₂O production results in large uncertainty about the estuarine contribution to the global N₂O budget. Incubation experiments with nitrogen stable isotope tracer were used to investigate the geochemical factors controlling N₂O production from denitrification in the Chesapeake Bay, the largest estuary in North America. The highest potential rates of water column N₂O production via denitrification (7.5±1.2&thinsp;nmol-N&thinsp;L⁻¹&thinsp;h⁻¹) were detected during summer anoxia, during which oxidized nitrogen species (nitrate and nitrite) were absent from the water column. At the top of the anoxic layer, N₂O production from denitrification was stimulated by addition of nitrate and nitrite. The relative contribution of nitrate and nitrite to N₂O production was positively correlated with the ratio of nitrate to nitrite concentrations. Increased oxygen availability, up to 7&thinsp;µmol&thinsp;L⁻¹ oxygen, inhibited both N₂O production and the reduction of nitrate to nitrite. In spring, high oxygen and low abundance of denitrifying microbes resulted in undetectable N₂O production from denitrification. Thus, decreasing the nitrogen input into the Chesapeake Bay has two potential impacts on the N₂O production: a lower availability of nitrogen substrates may mitigate short-term N₂O emissions during summer anoxia; and, in the long-run (timescale of years), eutrophication will be alleviated and subsequent reoxygenation of the bay will further inhibit N₂O production.</p>