Future rates of atmospheric N deposition have the potential to slow litter decay and increase the accumulation of soil organic matter by repressing the activity of lignolytic soil microorganisms. We investigated the relationship between soil biochemical characteristics and enzymatic responses in a series of sugar maple (Acer saccharum)-dominated forests that have been subjected to 16 yrs of chronic N deposition (ambient þ 3 g NO3 eN m2 yr1), in which litter decay has slowed and soil organic matter has accumulated in sandy spodosols. Cupric-oxide-extractable lignin-derived phenols were quantified to determine the presence, source, and relative oxidation state of lignin-like compounds under ambient and experimental N deposition. Pools of respired C and mineralized N, along with rate constants for these processes, were used to quantify biochemically labile substrate pools during a 16-week laboratory incubation. Extracellular enzymes mediating cellulose and lignin metabolism also were measured under ambient and experimental N deposition, and these values were compared with proxies for the relative oxidation of lignin in forest floor and surface mineral soil. Chronic N deposition had no influence on the pools or rate constants for respired C and mineralized N. Moreover, neither the total amount of extractable lignin (forest floor, P ¼ 0.260; mineral soil, P ¼ 0.479), nor the relative degree of lignin oxidation in the forest floor or mineral soil (forest floor P ¼ 0.680; mineral soil P ¼ 0.934) was influenced by experimental N deposition. Given their biochemical attributes, lignin-derived molecules in forest floor and mineral soil appear to originate from fine roots, rather than leaf litter. Under none of the studied circumstances was the presence or relative oxidation of lignin correlated with the activity of cellulolytic and lignolytic extracellular enzymes. Although chronic atmospheric N deposition has slowed litter decay and increased organic matter in our experiment, it had little effect on biochemical composition of lignin derived molecules in forest floor and surface mineral soil suggesting organic matter has accumulated by other means. Moreover, the specific dynamics of lignin phenol decay is decoupled from short-term organic matter accumulation under chronic N deposition in this ecosystem.