In past decades, environmental nitrogen fixation has been attributed almost exclusively to the action of enzymes in the well-studied molybdenum-dependent nitrogen fixation system. However, recent evidence has shown that nitrogen fixation by alternative pathways may be more frequent than previously suspected. In this study, the nitrogen fixation systems employed by lichen-symbiotic cyanobacteria were examined to determine whether their diazotrophy can be attributed, in part, to an alternative pathway. The mining of metagenomic data (generated through pyrosequencing) and PCR assays were used to determine which nitrogen-fixation systems are present in cyanobacteria from the genusNostocassociated with four samples from different geographical regions, representing different lichen-forming fungal species in the genusPeltigera. A metatranscriptomic sequence library from an additional specimen was examined to determine which genes associated with N2fixation are transcriptionally expressed. Results indicated that both the standard molybdenum-dependent system and an alternative vanadium-dependent system are present and actively transcribed in the lichen symbiosis. This study shows for the first time that an alternative system is utilized by cyanobacteria associated with fungi. The ability of lichen-associated cyanobacteria to switch between pathways could allow them to colonize a wider array of environments, including habitats characterized by low temperature and trace metal (e.g. molybdenum) availability. We discuss the implications of these findings for environmental studies that incorporate acetylene-reduction assay data. [ABSTRACT FROM AUTHOR]