Estimates of rates of organic matter degradation by bacterial sulfate reduction in aquifers are few, and all are obtained by indirect means. Here we present the first direct radiotracer measurements of in situ rates of sulfate reduction for an aquifer (Rømø, Denmark). Sulfate reduction occurs in distinct reaction zones within the aquifer at rates that vary greatly over short lateral distances. In situ rates of sulfate reduction are significantly higher than previous indirect estimates. In contrast, rates of sulfate reduction in aquifers are orders of magnitude lower than in marine and limmic environments, emphasizing the low reactivity of natural organic matter in aquifers.