Factors controlling the spatial distribution of benthic sulfate reduction (SR) were investigated at 3 stations [upper (UB), mid (MB) and lower bay (LB)] along the Chesapeake Bay (eastern USA) central channel from early spring through late fall, 1989 to 1994. Annual rates of 0 to 12 cm depth-integrated SR were 0.96, 9.62 and 6.33 mol S m-2 yr-1 for UB, MB and LB, respectively, as calculated from 35SO42- incubations. SR was carbon limited at UB, LB, and at the sediment surface at MB, and SO42- limited at depth at MB. Temperature explained 33 to 68% of the variability in annual rates, with an apparent influence on SR which increased in the seaward direction in surface sediments. We speculate that the enhanced response of SR to temperature in LB surface sediments was linked to seasonal variations in macrofaunal activity associated with temperature. Estimates of reduced-S burial indicated that only 4 to 8% of sulfur reduced annually was buried as Fe-S minerals at MB and LB, with the remainder presumably being reoxidized. In contrast, >50% of the sulfur reduced annually was buried at UB, due to comparatively low SR rates and the high concentration of reactive iron in the oligohaline region. SR mineralized 18 to 32% of the annual primary production. Our results indicate that organic quality may be more important than the absolute quantity of organic loading in dictating the magnitude of benthic SR rates along an estuarine gradient. Spatial trends in SR reflected the combined influence of deposited organic matter quality and quantity, SO42- availability, the presence or absence of benthic macrofauna, overlying water dissolved O2 conditions, reduced-S reoxidation dynamics, and iron-sulfide mineral formation.