Natural organic matter (NOM) composition affecting mercury (Hg) transport along a watershed transect were examined in the Lake 658 system at the Experimental Lakes Area, Canada. This watershed was dosed with an enriched stable isotope of Hg, allowing for distinction between recently deposited and historic Hg. Newly deposited Hg was not detected in significant quantities in upland flow or the lake, and occurred only in upper soil horizons, indicating that Hg has not reached steady state 8 years following deposition. Characterization of dissolved phase NOM was conducted by molecular weight fractionation, and analysis of absorbance properties and lignin phenols. Low molecular weight compounds were more mobile in the upland, while high molecular weight fractions contained more Hg. Spectral properties were not consistent predictors of Hg, but supported findings on molecular weight distribution. Source material composition, as indicated by lignin phenols, did not vary widely and was not correlated with Hg.