- At Site U1385, drilled during IODP Expedition 339 off the coast of Portugal on the continental slope, high-resolution sulfate concentration measurements in the pore fluids display non-steady-state behavior. At this site there is a zone of sulfate reduction in the uppermost seven meters of sediment, followed by a 38-meter interval where sulfate concentrations do not change, and finally sulfate concentrations are depleted to zero between 45 and 55 meters below seafloor. Below the sulfate minimum zone, there is abundant methane, suggesting that the lower sulfate consumption zone is coupled to anaerobic methane oxidation. We analyze pore water samples from IODP Site U1385 for sulfur and oxygen isotope ratios of dissolved sulfate, as well as the sulfur isotope composition of sedimentary pyrite. The sulfur isotopes in pore fluid sulfate display similar non-steady-state behavior similar to that of the sulfate concentrations, increasing over the uppermost zone of sulfate reduction and again over the lower zone of sulfate-driven anaerobic methane oxidation. The oxygen isotopes in sulfate increase to the ‘apparent equilibrium’ value in the uppermost zone of sulfate reduction and do not change further. Our calculations support the idea that sulfite to sulfide reduction is the limiting step in microbial sulfate reduction, and that the isotope fractionation expressed in the residual pore water sulfate pool is inversely proportional to the net sulfate reduction rate. The sulfur isotope composition of pyrite acquires one value in the uppermost sediments, which may be overprinted by a second value in the deeper sediments, possibly due to iron release during anaerobic methane oxidation or iron diffusion from a higher zone of bacterial iron reduction. Our results have implications for modeling the sulfur isotope composition of the pyrite burial flux in the global biogeochemical sulfur cycle.