Mercury (Hg) stable isotope fractionation has recently been developed as a tool in biogeochemistry. In this study, the extent of Hg stable isotope fractionation during reduction of ionic mercury [Hg(II)] by two Hg(II)-resistant strains, Bacillus cereus 5 and the thermophile Anoxybacillus sp. FB9 [which actively detoxify Hg(II) by the mer system] and a Hg(II)-sensitive metal-reducing anaerobe, Shewanella oneidensis MR-1 [which reduces Hg(II) at low concentrations], was investigated. In all cases, barring suppression of fractionation that is likely due to lower Hg(II) bioavailability, the Hg(II) remaining in the reactor became progressively enriched with heavy isotopes with time and underwent mass-dependent Rayleigh fractionation with alpha202/198 values of 1.0016 +/- 0.0004 (1 SD). Based on a multistep framework for the Hg(II) reduction pathways in the three strains, we constrain the processes that could contribute toward fractionation and suggest that for Hg(II)-resistant strains, reduction by mercuric reductase is the primary step causing fractionation. The proposed framework helps explain the variation in the extent of Hg stable isotope fractionation during microbial reduction of Hg(II), furthering the promise of Hg isotope ratios as a tool in determining the role of microbial Hg transformations in the environment.