A remarkable diversity of non-cultivated diazotrophs exists in soils suggesting that current models of N-fixation, based on cultivated strains, may not adequately explain the activity of these organisms in natural communities. Stable Isotope Probing (SIP) with 15N2 represents a powerful new tool that can be used to identify the functional capacities of non-cultivated diazotrophs in microbial communities. SIP has been used to examine diazotrophic community composition in soils incubated in the presence of 15N2 under different experimental conditions. During SIP experiments total N-fixation was assessed by measuring the incorporation of 15N into soil. N-fixation was greater in soils incubated under field conditions than in soils that were either saturated or soils from which oxygen was excluded. DNA was extracted from soils and subjected to centrifugation in CsCl gradients. Targeted CsCl fractions were subjected to secondary centrifugation in the presence of bis-benzimidazole in order to disentangle isotope incorporation from genome G + C content. The density of DNA from 15N containing fractions shifted to a greater degree in the presence of bis-benzimidazole than did DNA from unlabeled control fractions, consistent with 15N-incorporation. Amplification and cloning of 16S rRNA genes from 15N-containing fractions reveled three groups of bacteria that were significantly overrepresented relative to controls. One set of sequences belonged to the Rhizobiales, while the other two belonged to deeply divergent groups of non-cultivated bacteria within the Beta Proteobacteria and Actinobacteria respectively. Both of these novel groups are likely to represent novel orders within their respective phyla. A database search revealed 96 16S rRNA gene sequences that correspond to the novel actinobacterial lineage with >90% of those clones originating from soils or soil associated habitats. Thus, 15N-SIP has enabled the discovery of a widespread previously uncharacterized group of soil associated non-cultivated diazotrophs.