The preferential inorganic nitrogen source for the seagrass Zostera noltii was investigated in plants from Ria Formosa, South Portugal. Rates of ammonium and nitrate uptake were determined at different concentrations of these nutrients (5, 25 and 50 μm), supplied simultaneously (NH4NO3) or separately (KNO3 and NH4Cl). The activity of the enzymes nitrate reductase (NR) and glutamine synthetase (GS) was also assessed. The results showed that ammonium is the preferential inorganic nitrogen source for Z. noltii, but, in the absence of ammonium, the species also has a high nitrate uptake capacity. The simultaneous availability of both inorganic nitrogen forms enhanced the uptake rate of ammonium and decreased the uptake rate of nitrate compared to when only one of the nitrogen forms was supplied. The activity of both enzymes was much higher in the leaves than in the roots, highlighting the importance of the leaves as primary reducing sites in the nitrogen assimilation process.