The potential for microbially mediated anaerobic redox cycling of iron (Fe) was examined in a first-generation enrichment culture of freshwater wetland sediment microorganisms. Most probable number enumerations revealed the presence of significant populations of Fe(III)-reducing (approximately 10(8) cells ml(-1)) and Fe(II)-oxidizing, nitrate-reducing organisms (approximately 10(5) cells ml(-1)) in the freshwater sediment used to inoculate the enrichment cultures. Nitrate reduction commenced immediately following inoculation of acetate-containing (approximately 1 mM) medium with a small quantity (1% v/v) of wetland sediment, and resulted in the transient accumulation of NO(2)(-) and production of a mixture of gaseous end-products (N(2)O and N(2)) and NH(4)(+). Fe(III) oxide (high surface area goethite) reduction took place after NO(3)(-) was depleted and continued until all the acetate was utilized. Addition of NO(3)(-) after Fe(III) reduction ceased resulted in the immediate oxidation of Fe(II) coupled to reduction of NO(3)(-) to NH(4)(+). No significant NO(2)(-) accumulation was observed during nitrate-dependent Fe(II) oxidation. No Fe(II) oxidation occurred in pasteurized controls. Microbial community structure in the enrichment was monitored by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified 16S rDNA and reverse transcription polymerase chain reaction-amplified 16S rRNA, as well as by construction of 16S rDNA clone libraries for four different time points during the experiment. Strong similarities in dominant members of the microbial community were observed in the Fe(III) reduction and nitrate-dependent Fe(II) oxidation phases of the experiment, specifically the common presence of organisms closely related (>or= 95% sequence similarity) to the genera Geobacter and Dechloromonas. These results indicate that the wetland sediments contained organisms such as Geobacter sp. which are capable of both dissimilatory Fe(III) reduction and oxidation of Fe(II) with reduction of NO(3)(-) to NH(4)(+). Our findings suggest that microbially catalysed nitrate-dependent Fe(II) oxidation has the potential to contribute to a dynamic anaerobic Fe redox cycle in freshwater sediments.