1. We used two separate approaches to estimate ambient ammonium cycling in the north and south branches of Kings Creek, a prairie stream. Chamber experiments were conducted to determine ammonium uptake and mineralization rates associated with epilithic biofilms and filamentous algae collected from the streams. A series of short-term whole-stream ammonium addition experiments were also conducted to estimate the rate of uptake at ambient stream concentrations, based on the relationship between ammonium concentrations and uptake rates. 2. Chamber experiments were scaled up to whole-stream levels, resulting in ambient gross uptake estimates of 0.08 μg−2 s−1 for the north branch and 0.16 μg−2 s−1 for the south branch. The substrata-specific estimates of mineralization were higher than uptake in both streams. 3. Substrata-specific measurements indicated that ammonium uptake is higher in riffle habitats than in pools habitats. The results of the short-term ammonium addition experiments support these findings. 4. Short-term ammonium addition experiments show that uptake rates saturate with increasing ammonium concentrations. The observed saturation of uptake rates is consistent with a Michaelis–Menten relationship. 5. Scaled estimates of uptake from the chamber experiments were similar to estimates of ambient ammonium uptake based on the whole-stream experiments, and were comparable with previous estimates of ammonium uptake and mineralization made by using stable isotope tracer methods in Kings Creek.