Phosphorus (P) concentrations in sediments and in surface and interstitial water from three gravel bars in a large river (Garonne River, southern France) were measured daily, downstream of a wastewater treatment plant for a city of 740 000 inhabitants (Toulouse). Measurements were made of vertical hydraulic gradient (VHG), total dissolved phosphorus (TDP), soluble reactive phosphorus (SRP) and total phosphorus (TP) in water and of three extractable forms of phosphorus (water extractable, NaOH extractable and H2SO4 extractable) in hyporheic sediments from the gravel bars. Dissolved phosphorus was the major contributor to TP (74–79%) in both interstitial and surface waters on all sampling dates, and in most cases surface water P concentrations were significantly higher than interstitial concentrations. Hyporheic sediment TP concentrations ranged between 269 and 465 µg g−1 and were highest in fine sediment fractions. Acid-extractable P, a non-bioavailable form, represented at least 95% of sediment TP. A positive relationship was observed between VHG and TP in two of the gravel bars, with wells that were strongly downwelling having lower TP concentrations. These results suggest that in downwelling zones, hyporheic sediments can trap surface-derived dissolved P, and that much of this P becomes stored in refractory particulate forms. Bioavailable P is mainly present in dissolved form and only occupies a small fraction of total P, with particulate P comprising the majority of total P.