Fixed nitrogen is a limiting nutrient in many marine environments. Only a subset of the microbial community has the ability to fix dinitrogen gas (N 2). Here, we document the transcription of nitrogenase reductase subunit nifH in N 2-fixing bacteria in the dark suboxic and uppermost sulfidic layers of the northeast Black Sea. In shallower oxic waters, no N 2 fixation activity was detected using mRNA, and stable isotopes (δ 15 N and δ 18 O) of nitrate supported the lack of N 2 fixation in oxic waters. On 2 expeditions in 2007, sampling in the suboxic zone (O 2< 10 µM, H 2 S below detection) and in deeper sulfidic waters yielded mRNA transcripts of nifH, even though NH 4+ was 1-5 µM. Multiple phylogenetic groups expressed nifH. Three uncultured groups of Cluster III type transcripts were detected, as well as 2 groups of Cluster I type sequences related to known sulfur oxidizers in the ε-proteobacteria and Halorhodospira. The depth range where N 2 fixation was found was also the depth range of chemoautotrophic production, as determined by a maximum in suspended organic nitrogen concentrations and from 16S rRNA at these depths, which was dominated by known chemoautotrophs Sulfurimonas, SUP05, and BS-GSO2. We suggest chemoautotrophy and competition with chemoautotrophs for ammonium as reasons for N 2 fixation in the presence of ammonium. Profiles of N 2 gas unequivocally show the importance of N loss in the suboxic zone of the Black Sea; however, our data suggest a role for N 2 fixation. These results suggest that N cycling is seldom unidirectional.