Black layer has been associated with a severe decline in the quality of turf on putting greens. It was suggested that the black layer results from dissimilatory sulfate (SO42–) reduction. This study was done to determine if SO42– reduction occurs in an existing black layer. Radioactive 35SO42– was used to calculate the rate of SO42– reduction in intact soil cores taken from an existing black layer in a `Penncross' creeping bentgrass (Agrostis palustris Huds. `Penncross') putting green. When 10–3M35SO42– with a specific activity of 1.554 × 105 Bq·mg–1 SO42– was injected into a core it reduced to sulfide (35S2–) at a mean rate of 7.1 nmol sulfur (S)/cm3 soil/d. Injecting azide (N3–) or molybdate (MoO42–) at 10% w/v with the label reduced the rate of SO42– reduction to 0.03 and 0.01 nmol S/cm3 soil/d, respectively. The effect of N3– confirmed that reduction of SO42– was biological, while the effect of MoO42– confirmed that the entities responsible for the reductive cycling were sulfate-reducing bacteria (SRBs). This was the first proof that biological reduction of SO42– produces S2– in a black layer from a creeping bentgrass putting green. It was concluded that the respiration of indigenous SRBs was linked to development of this black layer. Thus, a key to successfully controlling black layer in putting greens must involve regulating the respiratory activities of SRBs.