Hydrological and biogeochemical processes in stream reaches impact the downstream transport of nutrients. The output from one stream reach becomes the input for the next, leading to serial processing along stream networks. The shape of the uptake-concentration curve for each reach indicates in-stream biological uptake of nutrient. Combined with physical retention due to hydrologic turnover, both biological and physical retention will control nutrient export downstream. We performed an instantaneous addition of conservative (chloride, Cl) and nonconservative nutrient (nitrate-nitrogen, NO3-N) tracers to ascertain the relative roles of physical and biological retention across four adjacent reaches along a 3744 m stream network in the Sawtooth Mountains, ID. Physical retention dominated total retention ranging from 15% to 58% across individual reaches and totaling 81% across the entire stream length. Within each reach, biological uptake was strongly controlled by nutrient concentration. We quantified continuous Michaelis-Menten (M-M) kinetic curves for each reach and determined that ambient uptake (Uamb) ranged from 19 to 58 μg m−2 min−1, maximum uptake (Umax) from 65 to 240 μg m−2 min−1, and half-saturation constants (Km) from 4.2 to 14.4 μg l−1 NO3-N. Biological retention capacity indicated by Umax decreased in a downstream direction. Although biological retention capacity decreased moving downstream, it did not decrease as much as physical retention, which led to biological retention comprising a larger portion of total retention at downstream reaches. We suggest that accurate assessment of total retention across stream reaches and stream networks requires quantification of physical retention and the concentration-dependent nature of biological uptake.