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Environmental flow releases have been advocated as a useful rehabilitation strategy for improving river condition but assessments of their success have typically focused on surface water quality and biota. In this study, we investigated the impacts of an environmental flow release on water temperature, conductivity, dissolved oxygen, and nitrate concentrations in surface and subsurface (hyporheic) water at upwelling and downwelling zones in three sites along the Hunter River, New South Wales, Australia. We hypothesised that the flow pulse would ’flush’ the sediments with oxygenated water, stimulating hyporheic microbial activity and nitrification, enhancing nitrate concentrations over time. Surface and subsurface samples were collected before, 7 days after, and 49 days after an environmental flow release of 5000 MI for a period of 3 days. No lasting effects on dissolved oxygen or conductivity were evident at most sites although dissolved oxygen declined over time at the downwelling site at Bowmans Crossing. At the downwelling zones at all sites, hyporheic nitrate concentrations declined initially following the release, but then rose or leveled off by Day 49. This initial drop in concentration was attributed to flushing of nitrate from the sediments. At two sites, nitrate concentrations had increased by Day 49 in the upwelling zones while at the third site, it fell significantly, associated with very low dissolved oxygen and likely reductive loss of nitrate. Electrical conductivity data indicate that potential inputs of agriculturally enriched groundwater may contribute to the nitrogen dynamics of the Hunter River. This study highlights the spatial heterogeneity that occurs in the hyporheic zone within and among sites of a regulated river, and emphasises the need for multiple-site surveys and an understanding of groundwater dynamics to assess physicochemical responses of the hyporheic zone to environmental flow releases.