Agricultural soils are heterogeneous environments in which conditions affecting microbial growth anddiversity fluctuate widely in space and time. In this study, the molecular ecology of the total bacterial andfree-living nitrogen-fixing communities in soils from the Nafferton Factorial Systems Comparison (NFSC)study in northeast England were examined. The field experiment was factorial in design, with organic versusconventional crop rotation, crop protection, and fertility management factors. Soils were sampled on threedates (March, June, and September) in 2007. Total RNA was extracted from all soil samples and reversetranscribed. Denaturing gradient gel electrophoresis (DGGE) and quantitative PCR (qPCR) were used toanalyze nifH and 16S rRNA genes in order to study free-living diazotrophs and the total bacterial community,respectively. Crop rotation was shown to have a significant effect on total bacterial diversity (and that offree-living N fixers) (P <0.001). On all three dates, nifH activity was higher in the conventional crop rotation.In contrast, qPCR analysis of free-living N fixers indicated significantly higher levels of activity in conventionallyfertilized plots in June (P 0.0324) and in plots with organic crop protection in September (P 0.0143). To our knowledge, the effects of organic and conventional farming systems on free-living diazotrophshave never been studied. An increased understanding of the impacts of management practices on free-living Nfixers could allow modifications in soil management practices to optimize the activity of these organisms.