ABSTRACT Biological nitrogen fixation is the primary supply of N to most ecosystems, yet there is considerable uncertainty about how N-fixing bacteria will respond to global change factors such as increasing atmospheric CO 2 and N deposition. Using the nifH gene as a molecular marker, we studied how the community structure of N-fixing soil bacteria from temperate pine, aspen, and sweet gum stands and a brackish tidal marsh responded to multiyear elevated CO 2 conditions. We also examined how N availability, specifically, N fertilization, interacted with elevated CO 2 to affect these communities in the temperate pine forest. Based on data from Sanger sequencing and quantitative PCR, the soil nifH composition in the three forest systems was dominated by species in the Geobacteraceae and, to a lesser extent, Alphaproteobacteria . The N-fixing-bacterial-community structure was subtly altered after 10 or more years of elevated atmospheric CO 2 , and the observed shifts differed in each biome. In the pine forest, N fertilization had a stronger effect on nifH community structure than elevated CO 2 and suppressed the diversity and abundance of N-fixing bacteria under elevated atmospheric CO 2 conditions. These results indicate that N-fixing bacteria have complex, interacting responses that will be important for understanding ecosystem productivity in a changing climate.