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Long-term effects of elevated atmospheric CO2 on the ammonia-oxidizing and denitrifying bacteria in a grassland soil were investigated to test whether a shift in abundance of these N-cycling microorganisms was responsible for enhanced N2O emissions under elevated atmospheric CO2. Soil samples (7.5 cm increments to 45 cm depth) were collected in 2008 from the University of Giessen Free Air Carbon dioxide Enrichment (GiFACE), a permanent grassland exposed to moderately elevated atmospheric CO2 (+20%) since 1998. GiFACE plots lay on a soil moisture gradient because of gradually changing depth to the underlying water table and labeled as the DRY block (furthest from water table), MED block (intermediate to water table), and WET block (nearest to water table). Mean N2O emissions measured since 1998 have been significantly higher under elevated CO2. This study sought to identify microbial and biochemical parameters that might explain higher N2O emissions under elevated CO2. Soil biochemical parameters [extractable organic carbon (EOC), dissolved organic nitrogen (DON), NH4+, NO3−], and abundances of genes encoding the key enzymes involved in ammonia oxidation (amoA) and denitrification (nirK, nirS, nosZ) depended more on soil depth and block (underlying soil moisture gradient) than on elevated CO2. Ammonia oxidation and denitrification gene abundances, relative abundances (ratios) of nirS to nirK, of nosZ to both nirS and to nirK, and of the measured soil biochemical properties DON and NO3− tended to be lower in elevated CO2 plots as compared with ambient plots in the MED and WET blocks while the DRY block exhibited an opposite trend. High N2O emissions under elevated CO2 in the MED and WET blocks correlated with lower nosZ to nirK ratios, suggesting that increased N2O emissions under elevated CO2 might be caused by a higher proportion of N2O-producing rather than N2O consuming (N2 producing) denitrifiers.