Soil phosphorus (P) concentrations typically are greater in surface soils compared with subsurface soils. Surface soils have a greater chance to interact with runoff leading to P transport to streams. The thin surface layer where P concentrates is referred to as the mixing layer denoting where water and chemicals mix during transport. The objective of this study was to evaluate the effect of hydrologic flow paths on soluble reactive phosphorus (SRP) loss at two temperatures. Laboratory flumes were built to simulate infiltration, return flow, saturation excess, and interflow, and subsequent interaction with the mixing layer. The sandy loam soil in the flumes was kept at saturation throughout all experiments, so that biochemical effects were normalized. Flow through the flumes was maintained at 3.6 mm/h for 24 to 99 h (at 6 and 25 degrees C) with water entering and exiting the flumes at different ports (to simulate different flow paths) or as low intensity rainfall. Experiments were performed with and without an artificially created P-enriched surface layer (5 mm thick, total P increased from 1010 mg/kg in the original soil to 2310 mg/kg by addition of dissolved phosphate). Results indicated that (i) SRP release was greater in soil with a mixing layer than in soil without a mixing layer; (ii) SRP release was greater during experiments at 25 degrees C than at 6 degrees C; (iii) at 25 degrees C, SRP release was greatest when water traversed the mixing layer in the upward direction (i.e., in return flow), and by flow parallel to the mixing layer (i.e., surface runoff); and (iv) at 6 degrees C, SRP release in subsurface flow following rainfall was slightly greater than in return flow and infiltration. Our results confirmed the presence of a variable, temperature-dependent desorption process when runoff water interacted with the mixing layer. Our findings have important implications for how different water flow paths in and over the soil interact with P in the soil, and what the ultimate concentration will be in runoff and interflow.