(.) The use of the C-13 : C-12 isotopic ratio ( delta C-13) of leaf- respired CO2 to trace carbon fluxes in plants and ecosystems is limited by little information on temporal variations in delta C-13 of leaf dark- respired CO2 ( delta C-13(r)) under field conditions. (.) Here, we explored variability in delta C-13(r) and its relationship to key respiratory substrates from collections of leaf dark- respired CO2, carbohydrate extractions and gas exchange measurements over 24- h periods in two Quercus canopies. (.) Throughout both canopies, delta C-13(r) became progressively C-13- enriched during the photoperiod, by up to 7%, then C-13- depleted at night relative to the photoperiod. This cycle could not be reconciled with delta C-13 of soluble sugars ( delta C-13(55)), starch ( delta C-13(st)), lipids ( delta C-13(l)), cellulose ( delta C-13(c)) or with calculated photosynthetic discrimination (Delta). However, photoperiod progressive enrichment in delta C-13(r) was correlated with cumulative carbon assimilation ( r(2) = 0.91). (.) We concluded that there is considerable short- term variation in delta C-13(r) in forest canopies, that it is consistent with current hypotheses for C-13 fractionation during leaf respiration, that leaf carbohydrates cannot be used as surrogates for delta C-13(r), and that diel changes in leaf carbohydrate status could be used to predict changes in delta C-13(r) empirically. (c) New Phytologist ( 2005).