Humic substances were extracted from the sediments of two small alpine lakes in the Colorado Front Range and characterized by three-dimensional fluorescence spectroscopy. The fluorescence index (FI), defined as the ratio of fluorescence emitted at 450 nm to that emitted at 500 nm from an excitation wavelength of 370 nm, was computed and thereafter compared to additional sediment proxies of recent environmental change. Stratigraphic changes in both sediment C:N ratios and diatom assemblages parallel those of Fl, together indicating pronounced increases in the contribution of autochthonous organic matter to lake sediments since the mid-20th century. This result is consistent with increased algal production attributable to nutrient enrichment, given thatthe region undergoes episodic nitrogen saturation in response to anthropogenic N emission. The validation of sediment Fl measurements through comparisons with independent methods demonstrates the utility of this technique for characterizing shifts in the provenance of lake sediment organic matter arising from changing environmental conditions.