Changes in the 13C discrimination of current leaf photosynthesis might have profound impacts on root respiratory substrates. Therefore, the aim of this study was (1) to refine a method for the isolation of root and leaf starch and soluble sugars (neutral fraction) for stable carbon isotope analysis and (2) to assess the short-term temporal variability of the C isotope composition (δ13C) of starch and of the neutral fraction of beech roots and leaves at different canopy heights. An existing method for isolating starch for stable C isotope analysis based on enzymatic hydrolysis was modified to account for the low starch content of the samples. This was achieved by removing the enzyme (α-amylase) by ultrafiltration after the hydrolysis, resulting in very low carbon blanks. The neutral fraction was separated from organic acids and cations by ion-exchange chromatography. An anion-exchange resin in the [HCO3]−-form was chosen that ensured high precision of C blanks. Beech leaves at 5, 10 and 20 m above the forest floor as well as roots were sampled six times during a day/night cycle in July 2003. δ13C values of bulk material, starch and the neutral fraction increased from the lower to the higher canopy with mean differences between 5 and 20 m of 3.8, 3.4 and 2.7‰ for the δ13C values of starch, neutral fraction and bulk foliage, respectively. The δ13C value of foliar starch increased from the morning to the afternoon and decreased during the night, but diurnal differences (up to 3.1‰) were only statistically significant for leaves sampled at 5 and 10 m height. In roots, no diurnal variation in the δ13C of starch was observed during the short time frame of one day and the δ13C of the neutral fraction did not differ between samples taken at 16:30 and 22:00. Calculated δ13C values of starch, which was mobilised during the night, were more positive than the total starch (all sampling times pooled) in leaves. Furthermore, the δ13C values of mobilised starch were approximately 5‰ more positive than that of the mobilised neutral fraction. Hence, the δ13C of potential sources for export from canopy leaves to roots varied considerably in their C isotope composition. Copyright © 2006 John Wiley & Sons, Ltd.