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Massive anthropogenic acceleration of the global nitrogen (N) cycle has stimulated interest in understanding the fate of excess N loading to aquatic ecosystems. Nitrate (NO3 −) is traditionally thought to be removed mainly by microbial respiratory denitrification coupled to carbon (C) oxidation, or through biomass assimilation. Alternatively, chemolithoautotrophic bacterial metabolism may remove NO3 − by coupling its reduction with the oxidation of sulfide to sulfate (SO4 2−). The NO3 − may be reduced to N2 or to NH4 +, a form of dissimilatory nitrate reduction to ammonium (DNRA). The objectives of this study were to investigate the importance of S oxidation as a NO3 − removal process across diverse freshwater streams, lakes, and wetlands in southwestern Michigan (USA). Simultaneous NO3 − removal and SO4 2− production were observed in situ using modified “push-pull” methods in nine streams, nine wetlands, and three lakes. The measured SO4 2− production can account for a significant fraction (25–40%) of the overall NO3 − removal. Addition of 15NO3 − and measurement of 15NH4 + production using the push–pull method revealed that DNRA was a potentially important process of NO3 − removal, particularly in wetland sediments. Enrichment cultures suggest that Thiomicrospira denitrificans may be one of the organisms responsible for this metabolism. These results indicate that NO3 −-driven SO4 2− production could be widespread and biogeochemically important in freshwater sediments. Removal of NO3 − by DNRA may not ameliorate problems such as eutrophication because the N remains bio-available. Additionally, if sulfur (S) pollution enhances NO3 − removal in freshwaters, then controls on N processing in landscapes subject to S and N pollution are more complex than previously appreciated. [ABSTRACT FROM AUTHOR]