Sulfate-reducing bacteria contribute considerably to the mineralization of petroleum hydrocarbons (PHC) in contaminated environments. Stable sulfur isotope fractionation during microbial. sulfate reduction was investigated in microcosm experiments with different cultures of sulfate-reducing bacteria for various initial sulfate concentrations using toluene as the sole carbon source. Experiments were conducted with the marine strain Desulfobacula toluolica, the fresh water strain PRTOL1, and an enrichment culture from a PHC-contaminated aquifer. Sulfate reduction rates ranged from 7 +/- 1 to 494 +/- 9 nmol cm(-3) d(-1), whereas specific sulfate reduction rates (sSRR) ranged from 8.9 X 10(-15) to 3.9 X 10(-13) +/- 9.2 X 10(-14) mol cell(-1) d(-1). Calculated enrichment factors (8) for the fractionation of stable sulfur isotopes during microbial sulfate reduction ranged from 19.8 +/- 0.9 to 46.9 +/- 2.1 parts per thousand. In general, values of epsilon and sSRR obtained in our experiments were similar to those reported previously for sulfate-reducing bacteria incubated with readily available carbon sources under optimal growth conditions. Moreover, we found no obvious correlation between epsilon and sSRR values when data from all our microcosm experiments were combined or when we combined our data with several previously published data sets. In contrast, epsilon values determined in our enrichment culture experiments (average 23.5 +/- 4.3 parts per thousand) agreed well with epsilon values determined in a recent field study performed in situ in a PHC-contarninated aquifer. Thus, results from this laboratory study provide valuable information on stable sulfur isotope fractionation during microbial sulfate reduction under conditions that more closely resemble those in PHC-contaminated environments, i.e., for a variety of sulfate concentrations, including low sulfate concentrations, and for a an important PHC-constituent (toluene) used as sole carbon source. Copyright (C) 2001 Elsevier Science Ltd.