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Activity of extracellular phosphatases (phosphomonoesterases) was measured in sandy streambed sediments of the Breitenbach, a small unpolluted upland stream in Central Germany. Fluorigenic 4-methylumbelliferyl phosphate served as a model substrate. Experiments were conducted using sediment cores in a laboratory simulation of diffuse groundwater discharge through the stream bed, a natural process occurring in the Breitenbach as well as many other streams.Streambed sediments contained high levels of particulate phosphorus, but concentrations of dissolved phosphorus in the interstitial water were 3 to 4 orders of magnitude lower. These interstitial concentrations were similar to those in the stream and groundwater. Extracellular phosphatase activity was high in the streambed sediments. These enzymes probably contribute significantly to the flux of phosphorus in sediment by hydrolyzing phosphomonoesters, making free phosphate available to the sediment microorganisms.Factors influencing the kinetic parameters Vmax (maximum activity) and apparent Km (enzyme affinity) of phosphatase were discharge rates of water through the sediment, water quality (ground-or stream water), and substrate (phosphomonoesters) as well as dissolved ortho-phosphate concentrations. Enzymes are supposed to be effective at limiting substrate concentrations, where, in this study, changes in discharge rates had little influence on rates of hydrolysis. Higher Vmax and lower Km values were found during percolation of groundwater through the sediment cores, compared with stream water. This indicates that rates of hydrolysis were higher with groundwater, both at substrate limitation and at substrate saturation. This was probably a consequence of the lower levels of dissolved ortho-phosphate in the groundwater.