Longitudinal and diel measurements of dual isotope composition (δ15N and δ18O) in nitrate (NO3-N) were made in the Ichetucknee River, a large (∼8 m3 s−1), entirely spring-fed river in North Florida, to determine whether isotopic variation can deconvolve assimilatory and dissimilatory removal. Comparing nitrate concentrations and isotope composition during the day and night we predicted (1) daytime declines in total fractionation due to low assimilatory fractionation and (2) diurnal variation in dual isotope coupling between 1:1 (assimilation) and 2:1 (denitrification). Five daytime longitudinal transects comprising 10 sampling stations showed consistent NO3-N removal (25–35% of inputs) and modest fractionation (15εtotal between −2 and −6‰, enriching the residual nitrate pool). Lower fractionation (by ∼1‰) during two nighttime transects, suggests higher fractionation due to assimilation than denitrification. Total fractionation was significantly negatively associated with discharge, input [NO3-N], N mass removal, and fractional water loss. Despite well-constrained mass balance estimates that denitrification dominated total N removal, isotope coupling was consistently 1:1, both for longitudinal and diel sampling. Hourly samples on two dates at the downstream location showed significant diel variation in concentration ([NO3-N] amplitude = 60 to 90 μg N L−1) and isotope composition (δ15N amplitude = −0.7‰ to −1.6‰). Total fractionation differed between day and night only on one date but estimated assimilatory fractionation assuming constant denitrification was highly variable and implausibly large (for N, 15ε = −2 to −25‰), suggesting that fractionation and removal due to denitrification is not diurnally constant. Pronounced counterclockwise hysteresis in the relationship between [NO3-N] andδ15N suggests diel variation in N isotope dynamics. Together, low fractionation, isotope versus concentration hysteresis, and consistent 1:1 isotope coupling suggests that denitrification is controlled by NO3− diffusion into the benthic sediments, the length of which is mediated by riverine oxygen dynamics. While using dual isotope behavior to deconvolve removal pathways was not possible, isotope measurements did yield valuable information about riverine N cycling and transformations.