Aims During the first days after harvest of Lolium perenne L., N remobilized from roots and stubble forms the main N source for regrowth. Low N uptake from the soil during this period may lead to N loss if N fertilizer is applied too soon. Furthermore, temporary N deprivation has been found to stimulate root growth. We therefore hypothesized that a strategic delay in N application after harvest may improve N-use efficiency of L. perenne grassland by increasing root biomass and reducing N loss. Methods In a laboratory and field experiment with L. perenne, we delayed N fertilizer application after harvest for 0,3,6,9 and 12 days, repeated this for up to six harvest cycles, and determined effects on herbage yield, herbage N uptake and root biomass. Results In both experiments, delaying N application for up to 12 days had no significant effect on root biomass or total herbage N uptake, but it significantly reduced total herbage yield in the laboratory experiment. Total yield tended to be highest when N application was delayed for 3 days. Two growth periods in the field experiment showed significantly higher N uptake when N application was delayed, possibly due to rainfallinduced N losses in the treatments with shorter delay. Conclusions Our results do not provide evidence that delaying N application improves N-use efficiency of L. perenne grassland by increasing root biomass. However, strategic timing of N fertilizer application based on rainfall forecasts could contribute to improve N-use efficiency by reducing N losses from leaching and denitrification.