We conducted a series of stepwise NO₃⁻ additions to investigate the response of NO₃⁻ uptake to short-term (acute) changes in N concentration in 3 prairie streams. Observed NO₃⁻ uptake rates increased with short-term elevations in NO₃⁻ concentration and were consistent with linear and Michaelis–Menten kinetics models. We compiled these data with uptake rates from additional published studies to calculate robust estimates of N uptake kinetics for prairie streams. Half-saturation coefficients based on compiled data were 6.7 µg/L for NH₄ and 67 µg/L for NO₃⁻-N. This difference in half-saturation coefficients suggests that NH₄ is more efficiently assimilated than NO₃⁻, indicating a preference for NH₄ as an N source. Similarly, ambient concentrations of NH₄ and NO₃⁻ were less than their respective half-saturation coefficients, and aerial uptake rates were generally <5% of the maximum, suggesting severe limitation of N uptake at ambient conditions. The observed pattern of uptake kinetics suggests that physiological constraints limit biotic N uptake in these low-N streams and contrasts with the pattern of uptake observed in streams with chronically elevated ambient NO₃⁻ concentrations.