Background/Question/Methods Nitrogen (N) and phosphorus (P) limit growth of both plants and microorganisms in all ecosystems. The microbial extracellular enzyme activity that supports soil fertility by releasing plant available nutrients is expressed as a response to nutrient limitation. Consequently, chronic N fertilization can cause losses of soil enzyme activity associated with competitive dominance of fast-growing soil microorganisms (copiotrophic hypothesis). However, in a circumneutral soil with low P availability, such as the tallgrass prairie at Konza Prairie Biological Station (Manhattan, KS), N fertilization might exacerbate P limitation and thus increase enzyme activity. If soil enzyme activity is lost due to chronic fertilization, similar levels of aboveground net primary production (ANPP) may not be sustained without supplemental fertilizer. To assess whether the copiotrophic hypothesis was supported in this field context, strips of burned or unburned blocks of prairie were fertilized annually with N, P, or N and P (NP), or were not fertilized (control) with 4 replicate plots for each combined field treatment. In 2015, soils from each plot were collected monthly starting in January to measure carbon (C)-, N-, and P-acquiring extracellular enzyme activities (EEAs) and bacterial and archaeal community composition. ANPP was also estimated at the end of the growing season. Results/Conclusions EEAs should decline in chronically fertilized prairie soils (i.e., copiotrophic hypothesis). However, cellulolytic enzyme activities (beta-glucosidase [BG] and cellobiohydrolase [CBH]) didn’t decline after N-fertilization. While N-acetylglucosaminidase (NAG) and leucyl aminopeptidase (LAP) activities decreased from N fertilization (P<0.01), phosphatase (PHOS) activity was higher in N-fertilized prairies (P<0.05) and didn’t change with P-fertilization.Together, the BG:(NAG+LAP) and (NAG+LAP):PHOS ratios, indicative of microbial C:N and N:P demands, respectively, show lower N demand (P<0.001) and higher P demand (P<0.001) by microbes in N-rich prairies. In further contrast to the copiotrophic hypothesis, unburned prairies, with more available N, had higher BG and CBH activities than burned prairies (P<0.05), but did not have higher PHOS activity. Also, grass ANPP was higher in the burned prairies (P<0.001), especially when fertilized with N (P<0.001). However, soil bacterial and archaeal community compositions did shift towards putative copiotrophic dominance in chronically N fertilized prairie soils. We found limited support for the enzyme feedback aspect of the copiotrophic hypothesis, likely due to concurrent increases in plant growth and C input, which causes relative nutrient limitation to persist despite compositional shifts in soil microbial communities.