<p>Extracellular enzymes that catalyze the hydrolysis of proteinaceous and chitinaceous forms of organic nitrogen (N) inputs into N-bearing monomers limit downstream N mineralization, though depolymerization rates are also modulated by organic N chemistry and soil fertility status. We evaluated the short-term (28 d) sensitivity of N-hydrolytic enzyme activities and N pool concentrations to chitinaceous (chitin, chitosan, cricket, mealworm) and proteinaceous (soy, wheat, casein) substrate additions to two soils under contrasting long-term (145 y) fertility management practices that differed markedly in nutrient status and organic matter content. We found that chitin and chitosan additions increased chitinase activities substantially in both soils (&gt; 7-fold), but unexpectedly, chitinaceous substrates also increased protease (chitin) and aminopeptidase (chitin and mealworm) activities in the fertilized soil under a diverse crop rotation with fertilization (high fertility soil) but not in the unfertilized soil under continuous maize (low fertility soil). Although proteinaceous substrates consistently decreased aminopeptidase activities in both soils, large magnitude increases in downstream free amino acid-N and ammonium-N (low fertility soil) and nitrate-N (high fertility soil) indicated amino acid production and mineralization likely occurred shortly after substrate additions (&lt; 28 d). In addition to short-term augmentation of hydrolytic soil enzyme activities and downstream N pools, most substrates increased soil respiration more in the low versus high fertility soil– without concurrent increases in microbial biomass carbon or N. Thus, our study demonstrates that long-term management practices can yield metabolically distinct soils that exhibit divergent, and often unexpected, responses to organic N additions.</p>