The natural 13C/12C isotope composition (δ13C) of plants and organic compounds within plant organs is a powerful tool to understand carbon allocation patterns and the regulation of photosynthetic or respiratory metabolism. However, many enzymatic fractionations are currently unknown, thus impeding our understanding of carbon trafficking pathways within plant cells. One of them is the 12C/13C isotope effect associated with invertases (EC 184.108.40.206) that are cornerstone enzymes for Suc metabolism and translocation in plants. Another conundrum of isotopic plant biology is the need to measure accurately the specific δ13C of individual carbohydrates. Here, we examined two complementary methods for measuring the δ13C value of sucrose, glucose and fructose, that is, off-line high-performance liquid chromatography (HPLC) purification followed by elemental analysis and isotope ratio mass spectrometry (EA-IRMS) analysis, and gas chromatography-combustion (GC-C)-IRMS. We also used these methods to determine the in vitro12C/13C isotope effect associated with the yeast invertase. Our results show that, although providing more variable values than HPLC∼EA-IRMS, and being sensitive to derivatization conditions, the GC-C-IRMS method gives reliable results. When applied to the invertase reaction, both methods indicate that the 12C/13C isotope effect is rather small and it is not affected by the use of heavy water (D2O). Copyright © 2009 John Wiley & Sons, Ltd.