The aim of the study was to evaluate the impact of defoliation intensity, defoliation frequency, and interactions with N supply on N uptake, N mobilization from and N allocation to roots, adult leaves, and growing leaves. Plants of Lolium perenne were grown under two contrasted N regimes. Defoliation intensity treatments consisted of a range of percentage leaf area removal (0, 25, 50, 75, or 100%). These treatments were applied in parallel to a set of plants previously undefoliated, and to a second set of plants which had been defoliated several times at a constant height. A N-15 tracer technique was used to quantify N uptake, mobilization, and allocation over a 7 d period. A significant reduction in plant N uptake was observed with the removal of more than 75% of lamina area, but only with high N supply. As defoliation intensity increased, the amount of N taken up and subsequently allocated to growing leaves during the labelling period was maintained at the expense of N allocation to roots and adult leaves. Increasing defoliation intensity increased the relative contribution of roots supplying mobilized N to growing leaves and decreased the relative contribution of adult leaves. Defoliation frequency did not substantially alter N uptake, mobilization, and allocation between roots, adult and growing leaves on a plant basis. However, tiller number per plant was largely increased under repeated defoliation, hence indicating that allocation and mobilization of N to growing leaves, on the basis of individual tillers, was decreased by defoliation frequency.