• In order to test whether xylem-transported carbohydrates are a potential source for isoprene biosynthesis, [U- 13 C]-labelled α- d -glucose was fed via cut ends of stems into the xylem of Quercus robur seedlings and the incorporation of 13 C into isoprene emitted was studied. Emission of 13 C-labelled isoprene was monitored in real time by proton-transfer-reaction mass spectrometry (PTR-MS).• A rapid incorporation of 13 C from xylem-fed glucose into single (mass 70) and double (mass 71) 13 C-labelled isoprene molecules was observed after a lag phase of approx. 5–10 min. This incorporation was temperature dependent and was highest (up to 13% 13 C of total carbon emitted as isoprene) at the temperature optimum of isoprene emission (40–42°C), when net assimilation was strongly reduced.• Fast dark-to-light transitions led to a strong single or double 13 C-labelling of isoprene from xylem-fed [U-13C]glucose. During a period of 10–15 min up to 86% of all isoprene molecules became single or double 13 C-labelled, resulting in a 13 C-portion of up to 27% of total carbon emitted as isoprene.• The results provide evidence that xylem-transported glucose or its degradation products can potentially be used as additional precursors for isoprene biosynthesis and that this carbon source becomes more important under conditions of limited photosynthesis.