Based on phylogenetic analysis of clones retrieved from two nifH gene clone libraries that were created using cDNA from suboxic sediment samples obtained from areas densely vegetated with the high-salt marsh plant Spartina patens, a primer set was designed to target nitrogen-fixing bacteria with sequence similarities to members of the epsilon subclass of Proteobacteria. Nested PCR, denaturing gel electrophoresis, and subsequent sequence analysis of reamplified fragments confirmed the specificity of the primer set by retrieving nifH sequences of only putative members of the epsilon subclass of Proteobacteria, all of which were characterized by a highly divergent 27- or 36-bp insertion in both DNA and cDNA.