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Along a single stream riffle, there is a typical flow pattern in which surface water enters the hyporheic zone in a downwelling zone at the head of the riffle and hyporheic water returns to the stream surface in an upwelling zone at the tail of the riffle. Distinct patterns of physical and chemical conditions in the hyporheic zone are likely to determine patterns of microbial activity and occurrence of hyporheic fauna. Interstitial water and core samples were taken at three depths in the downwelling and upwelling zones of a single riffle in the Speed River, Southern Ontario, Canada. Physical and chemical characteristics of the hyporheic water, bacterial density, protein content, detritus content and faunal composition of the hyporheic sediment were analysed. The downwelling and upwelling zones differed significantly in temperature, pH, redox potential, dissolved oxygen and nitrate with significant positive correlations occurring among the latter three. There were no differences in bacterial density or detritus content between the two zones nor between depths in either zone, but protein content, considered to be a measure of biofilm biomass, was significantly higher in the downwelling zone. Total density of hyporheic fauna and the number of taxa decreased with increasing depth in both upwelling and downwelling zones, and were positively correlated with surface water characteristics (oxygen, temperature and nitrate), sediment protein content and detritus; however, only a weak correlation was found with zone. The composition of taxa differed between the two zones, and faunal distribution was correlated with dissolved oxygen, detritus, protein content and depth.