A common method to quantify stream nutrient uptake length is to enrich the stream at an upstream point and monitor the downstream decline in the concentration of the added nutrient. However, increasing the nutrient concentration of the stream alters nutrient uptake, and uptake length quantified using this amendment approach does not necessarily reflect uptake length under ambient conditions. We conducted a series of 15NO3 - tracer and 14NO3- nutrient amendment experiments to (1) assess the overestimation of amendment-derived uptake length in streams spanning a gradient of ambient nitrogen concentration, and (2) evaluate a technique to estimate ambient uptake length using data from multiple amendments conducted at different concentrations. Single amendment-derived uptake lengths were consistently longer than ambient uptake lengths quantified using the stable-isotope tracer. The ratio of single amendment-derived to ambient uptake length ranged more than sixfold across streams but was not related to ambient stream water nitrogen concentration or amendment concentration. Estimated ambient uptake length calculated from the multiple amendment technique was a better predictor of ambient uptake length than single amendment-derived uptake length in three of five experiments. However, with this technique, calculated uptake length was negative in two streams. Our results suggest that nitrogen limitation was weak in these two streams and that the multiple amendment technique may be ineffective in streams where the nutrient of study is not limiting.