Biofilms can methylate mercury (Hg) at higher rates than unattached bacteria and are increasingly recognized as important Hg methylation sites in the environment. Our previous study showed that methylation rates in biofilm cultures were up to 1 order of magnitude greater than those in planktonic cultures of a sulfate-reducing bacterium. To probe whether the differential Hg methylation rates resulted from metabolic differences between these two cultures, Hg methylation assays following molybdate or chloroform inhibition (a specific inhibitor of the acetyl-CoA pathway) were conducted on biofilm and planktonic cultures of Desulfovibrio desulfuricans strains M8 and ND132. Molybdate was as effective in inhibiting Hg methylation as well as growth in both planktonic and biofilm cultures. The addition of chloroform only impacted Hg methylation in biofilm cultures, suggesting that different pathways are used for methylation in biofilm compared to planktonic cultures. To investigate this further, expression of the cooS gene, which encodes for carbon monoxide dehydrogenase, a key enzyme in the acetyl-CoA pathway, was compared in biofilm and planktonic cultures of ND132. Biofilm cultures showed up to 4 times higher expression of cooS than planktonic cultures. On the basis of these results, the acetyl-CoA pathway appears to play an important role in methylation in biofilm cultures of this organism, possibly by supplying the methyl group to Hg methylating enzymes; methylation in planktonic cultures appears to be independent of this pathway. This observation has important implications, particularly in developing reliable models to predict Hg methylation rates in different environments and perhaps eventually in being able to control this undesirable chemical transformation.