An integrated field and laboratory study was conducted to quantify the effect of environmental determinants on the activity of sulfate reducers in a freshwater aquifer contaminated with petroleum hydrocarbons (PHC). Within the contaminated zone, PHC-supported in situ sulfate reduction rates varied from 11.58 ± 3.12 to 636 ± 53 nmol cm -3 d -1 and a linear increase (R 2 =0.98) in reduction rate was observed with increasing in situ sulfate concentrations suggesting sulfate limitation. Half-saturation concentration (K s ) for sulfate reduction coupled to PHC mineralization was determined for the first time. At two different sites within the aquifer, maximum sulfate reduction rate under non-limiting conditions (R max ) was 5,000 nmol cm -3 d -1 , whereas the retrieved K s values were 3.5 and 7.5 mM, respectively. The K s values are the highest ever reported from a natural environment. Furthermore, the K s values were significantly higher than in situ sulfate concentrations confirming sulfate limited growth. On addition of lactate and formate, sulfate reduction rate increased indicating that reactivity and bioavailability of organic substrate may also have played a role in rate inhibition in certain parts of the aquifer. Experiments with sulfide amendments show statistically minor decrease in sulfate reduction rates on addition of sulfide and analogous increase in sulfide toxicity with increasing sulfide concentrations (0.5-10 mM) was not apparent.