1. An increase in human population and associated changes in land use have caused an increase in groundwater nitrate concentrations throughout central Florida. Within the region, this nitrate-laden groundwater returns to the surface via numerous large springs that serve as the origin of flow for many coastal streams and rivers. These rivers can exhibit strong nitrate gradients because of the high nutrient uptake potential of the rivers. 2. We hypothesised that downstream declines in nitrate concentrations would be manifested spatially as increases in the delta N-15 of the residual pool of nitrate, macrophytes and periphyton as a consequence of isotopic fractionation associated with preferential use of (NO3-)-N-14. This hypothesis was tested in two spring-fed river systems, the Chassahowitzka and Homosassa rivers, along Florida's central Gulf of Mexico coast. 3. In general, delta N-15 values of nitrate, macrophytes and periphyton increased with decreasing fraction of nitrate remaining in each of the two study systems. The fractionation associated with nitrate uptake by macrophytes and associated periphyton was determined from the relationship between delta N-15 of both constituents of the macrophyte community and the fraction of nitrate removed from the system. Values for fractionation by macrophytes and periphyton ranged from 1.9 parts per thousand to 3.6 parts per thousand and from 0.7 parts per thousand to 2.5 parts per thousand, respectively.