Anthropogenic activities increase rates of N input to the environment, and loss of this N is controlled by several factors, including denitrification. Streams are the initial receptors of terrestrial N, but the extent to which variability in stream denitrification rates are related to differences in microbial community structure are largely unexplored. In our study, the rate of denitrification and taxonomic and functional gene diversity and abundance were examined in 3 Indiana (USA) streams with differing amounts of watershed agriculture. Taxonomic and functional gene diversity were measured using terminal restriction length polymorphisms of the 16S ribosomal ribonucleic acid (rRNA) and nitrous oxide reductase (nosZ) genes, and abundance was examined using quantitative polymerase chain reaction (Q-PCR) and total direct cell counts. As expected, streams with highest amounts of watershed agriculture had highest NO₃⁻ concentrations and highest sediment organic matter (OM) content leading to higher denitrification rates. Overall, denitrification rates were controlled primarily by sediment OM content and secondarily by nosZ abundance and nosZ terminal restriction fragment (T-RF) number. However, total bacterial numbers were not related to peaks in denitrification rate. The 2 sites with the most substantial differences in watershed agriculture, NO₃⁻ concentrations, and sediment OM content also had the largest differences in both nosZ abundance and nosZ gene profiles. Overall, our results suggest that denitrification rates in agricultural streams are influenced by a combination of environmental variables (primarily benthic OM and NO₃⁻ concentrations) and microbial community composition.