We investigated microbial methylmercury (CH(3)Hg) production in sediments from the South River (SR), VA, an ecosystem contaminated with industrial mercury (Hg). Potential Hg methylation rates in samples collected at nine sites were low in late spring and significantly higher in late summer. Demethylation of (14)CH(3)Hg was dominated by (14)CH(4) production in spring, but switched to producing mostly (14)CO(2) in the summer. Fine-grained sediments originating from the erosion of river banks had the highest CH(3)Hg concentrations and were potential hot spots for both methylation and demethylation activities. Sequencing of 16S rRNA genes of cDNA recovered from sediment RNA extracts indicated that at least three groups of sulfate-reducing bacteria (SRB) and one group of iron-reducing bacteria (IRB), potential Hg methylators, were active in SR sediments. SRB were confirmed as a methylating guild by amendment experiments showing significant sulfate stimulation and molybdate inhibition of methylation in SR sediments. The addition of low levels of amorphous iron(III) oxyhydroxide significantly stimulated methylation rates, suggesting a role for IRB in CH(3)Hg synthesis. Overall, our studies suggest that coexisting SRB and IRB populations in river sediments contribute to Hg methylation, possibly by temporally and spatially separated processes.