The eastern Mediterranean Sea is one of the most extreme oligotrophic oceanic regions on earth in terms of nutrient concentrations and primary productivity. Nitrogen fixation has been suggested to contribute to the high N : P molar ratios of ∼28:1 found in this region. Surprisingly, no molecular biological work has been performed in situ to assess whether N2 fixation genes actually occur in the eastern Mediterranean Sea, or to determine which organisms are responsible for this process. In this study, we examined the presence and expression of nitrogenase genes (nifH) in the upper water layer of the eastern Mediterranean. Clone libraries constructed from both DNA and reverse-transcribed PCR-amplified mRNA were examined and compared. We observed different nifH genes from diverse microbial groups, such as Cyanobacteria, Proteobacteria and methanogenic Archaea. Interestingly, numerous phylotypes were observed in coastal stations at the DNA level but none were active. However, in far offshore stations, the phylotypes observed at the DNA level were the ones that were actually active. Our preliminary study revealed diverse diazotrophs that possess and express nifH genes, which may support N2 fixation in the eastern Mediterranean Sea.