In situ 13C/12C isotopic labelling was conducted in field‐grown beech (Fagus sylvatica) twigs to study carbon respiration and allocation. This was achieved with a portable gas‐exchange open system coupled to an external chamber. This method allowed us to subject leafy twigs to CO2 with a constant carbon isotope composition (δ13C of −51.2‰) in an open system in the field. The labelling was done during the whole light period at two different dates (in June 2002 and October 2003). The δ13C values of respiratory metabolites and CO2 that is subsequently respired during the night were measured. It was found that night‐respired CO2 is not completely labelled (only ca. 58% and 27% of new carbon is found in respired CO2 immediately after the labelling in June 2002 and October 2003, respectively) and the labelling level progressively disappeared during the next day. It is concluded that the carbon respired by beech leaves after illumination was supplied by a mixture of carbon sources in which current carbohydrates were not the only contributors. In addition, as has been found in herbaceous plants, isotopic data before labelling showed that carbon isotope discrimination favoring the 13C isotope occurred during the night respiration of beech leaves.