Batch culture experiments were performed with 32 sulfate-reducing prokaryotes to explore the diversity in sulfur isotope fractionation during dissimilatory sulfate-reduction by pure cultures. The selected strains reflect the phylogenetic and physiologic diversity of presently known sulfate reducers and were isolated from a broad range of natural marine and freshwater habitats. Experimental conditions were designed to achieve optimum growth conditions with respect to electron donors, salinity, temperature, and pH. Under these optimized conditions, experimental fractionation factors ranged from 2.0 permil to 42.0 permil. Salinity, incubation temperature, pH, and phylogeny had no systematic effect on the sulfur isotope fractionation. There was no correlation between isotope fractionation and sulfate reduction rate. The type of dissimilatory bisulfite reductase also had no effect on fractionation. Sulfate reducers that oxidized the carbon source completely to carbon dioxide showed greater fractionations than sulfate reducers where acetate is the final product of carbon oxidation. Different metabolic pathways and variable regulation of sulfate transport across the cell membrane all potentially affect isotope fractionation. Previous models that explained fractionation only in terms of sulfate reduction rates appear to be oversimplifying. The species-specific physiology of each sulfate reducer needs to be taken into account to understand the regulation of sulfur isotope fractionation during dissimilatory sulfate reduction.