Recent publications suggest that in large watersheds a large proportion (20 to 70%) of the net nitrogen input to the aquatic environment may be removed during water’s downstream path towards the outlet. In small watersheds there are few to no evaluations of the importance of in-stream processes in the overall nitrogen budget due in part to the lack of simple and robust methods for measuring in-stream nitrate uptake. We propose a comparative study for laboratory and field measurements on open mesocosms, 30 cm in diameter. Nitrate uptake was evaluated from the kinetics of disappearance of nitrate in water overlying undisturbed sediment cores, both in the laboratory and in-situ. Using both laboratory and in-situ mesocosms was an effort to determine whether the methods were comparable and applicable on a routine basis. Nitrate disappearance kinetics in the laboratory yielded expected results, which are that nitrate uptake rates are linearly correlated to nitrate concentration in the water column. In such conditions, our results show that the potential for nitrate uptake in streams can be calculated from the mass transfer coefficient. Our comparative study shows that incubations conducted in the laboratory intrinsically limit hyporheic exchanges, associated with small water level fluctuations, which may in fact prevail in the field as the in-situ incubations have shown. In-situ experiments yielded unexpected results, such as the succession of disappearance and gain of nitrate in the mesocosms through time. This was attributed to hyporheic exchanges, both natural and artificially induced by the design of the mesocosms. Both methods should be considered for future nitrate uptake studies as they yield complementary results, provided that some simple changes are made in the design of in-situ mesocosms.