Previous studies have indicated that an increased asparagine to glutamine ratio (Asn : Gln) occurs in the xylem fluid of Lolium perenne 24 h after defoliation. However, the absolute changes in Asn and Gln leading to the increased Asn : Gln ratio are unknown. The present study tested the hypotheses that: (1) defoliation-induced changes in xylem amino acid composition occur in L. perenne within the first 24 h following defoliation, irrespective of phasing with respect to the diurnal light/dark cycle; and (2) the increase in Asn : Gln ratio in the xylem fluid of L. perenne following defoliation is due to an increase in Asn content. Plants of L. perenne L. ‘Aurora’ were grown in flowing solution culture for 40 d. Plants were then either left intact, defoliated at the end of the light period or defoliated at the end of the dark period. 15N-labelled NO3– was supplied following defoliation to discriminate between the recovery of N absorbed prior to, and following, defoliation. Xylem samples were collected over the subsequent 24 h period with amino acids speciated by GC-MS. There was support for the first hypothesis: increased Asn : Gln ratios occurred within the first 24 h, irrespective of the phasing of defoliation with respect to light/dark cycles. The second hypothesis was not supported: the concentration of all amino acids in the xylem exudate declined after defoliation, and the increased Asn : Gln ratio was accounted for by a disproportionately large reduction in Gln levels. Low concentrations of amino acids in the xylem of defoliated plants precluded accurate discrimination of their nitrogen content into pre- and post-defoliation sources.