Denitrification removes large amounts of reactive nitrogen (N) from ecosystems via reduction of nitrate to dinitrogen gas. In aquatic ecosystems, the influences of terrestrial and aquatic sources of organic matter (OM) on denitrification are potentially complex. Terrestrially-derived OM is often less labile than autochthonous OM; it may inhibit denitrification directly via biochemical mechanisms; and it may indirectly inhibit denitrification by reducing light availability to—and thus OM exudation by—aquatic primary producers. Using a natural dissolved OM gradient among rivers of northern Florida, I investigated these mechanisms using laboratory denitrification assays subjected to factorial amendments of NO3- and dextrose, humic acid dosing, and cross-incubations of sediments and water. Results indicated that C-limitation increased with DOC concentrations, consistent with the indirect inhibition hypothesis. Blackwater neither depressed nor stimulated denitrification rates, indicating that this DOC neither directly inhibits nor acts as a usable OM source for denitrifiers.