Orchard grass (Dactylis glomerata) is a common perennial grass that is frequently found in woodland edges, rough grassland, meadows and permanent pastures. In the UK and continental Europe, orchard grass, together with other native species, including fescues, occupies around 30% of pastoral land. Polyphenol oxidases are enzymes that cause enzymatic browning by conversion of natural phenolic compounds, such as monophenols to o-dihydroxyaryl compounds and subsequent oxidation to o-quinones . These highly electrophilic species bind rapidly to nucleophilic sites on other compounds (e.g. phenols, amino-acids and proteins) and condense with phenols to form brown-, black- or red-colored polymers, associated with the undesired discoloration of fruit and vegetables. Recently, high levels of PPO activity have been reported in orchard grass ; however, to date, no endogenous substrates have been identified. In the present study, we report the isolation and structural elucidation of PPO substrates in this species. The free phenol fraction was extracted, separated by reverse-phase chromatography and six potential substrates, including two novel hydroxycinnamate esters (2-O-caffeoylisocitric acid 6-methyl ester and 2-O-caffeoylthreonic acid), were identified by UV spectrometry, electrospray ionisation-tandem mass spectrometry (LC-ESI-MSn) and 1D and 2D NMR analyses (1H NMR, 13C NMR, DEPT, COSY, HMQC and HMBC). Furthermore, three caffeoylquinic acids (3-CQA, 4-CQA and 5-CQA) were identified by comparison of their spectral data (ESI-MS) with those of known compounds and literature data. Five of the isolated compounds (i.e. chlorogenic acid, 2-O-caffeoylisocitric acid, 2-O-caffeoylisocitric acid 6-methyl ester, 2-O-caffeoylthreonic acid and 2-O-caffeoylhydroxycitric acid) were demonstrated to be substrates for orchard grass PPO.