ABSTRACT Natural isotopic abundances of N (d15N) in organic matter are used to examine the trophic relations in river and hyporheic habitats of the Flathead River, Montana. The d15N of biofilms and Plecopteran-dominated food webs are compared for river and hyporheic waters. We examine the concept that the isotope ratio of an animal undergoes both the food source effect and in vivo metabolic effects. Metabolic effects for nitrogen isotopes in animal tissues relates to isotopic fractionation, which exhibits progressive enrichment of the d15N in body tissues at higher trophic levels. These ∇ d15N enrichments are in excess of isotopic abundances of food sources. Comparisons show greater enrichment for the consumer trophic levels of the hyporheic than river channel habitat. The d15N contents of the hyporheic food webs suggest influences of different concentrations and d15N contents of NO3 and NH4. These inorganic conditions may be controlled by physical mixing of various water masses, Ntransformations associated with biofilm microbiota, and feedbacks of NH4 through excretion by animals. Our observations indicate that stable isotopes may be powerful tools for determining nitrogen source and process information in hyporheic habitats.