Mercury (Hg) contamination is widespread in remote areas of the northeastern United States. Forested uplands have accumulated a large reservoir of Hg in soil from decades of elevated anthropogenic deposition that can be released episodically to stream water during high flows. The objective of this study was to evaluate spatial and temporal variations in stream water Hg species and organic matter fractions over a range of hydrologic conditions in three forested upland watersheds (United States). Mercury and organic matter concentrations increased with discharge at all three sites; however, the partitioning of Hg fractions (dissolved versus particulate) differed among sites and seasons. Associated with increased discharge, flow paths shifted from mineral soil under base flow to upper soil horizons. As flow paths shifted, greater concentrations of dissolved organic carbon (DOC) richer in aromatic substances were flushed from upper soil horizons to stream water. The hydrophobic organic matter associated with humic material from upper soils appears to have had a greater capacity to bind Hg. Because of the strong correlation between Hg and DOC, we hypothesize that there was a concurrent shift in the source of Hg with DOC from lower mineral soil to upper soil horizons. Our study suggests that stream discharge is an effective predictor of dissolved total Hg flux.