Carbon isotope effects were investigated for the reaction catalyzed by the glycine decarboxylase complex (GDC; EC 188.8.131.52). Mitochondria isolated from leaves of pea (Pisum sativum L.) and spinach (Spinacia oleracea L.) were incubated with glycine, and the CO2 evolved was analyzed for the carbon isotope ratio (δ13C). Within the range of parameters tested (temperature, pH, combination of cofactors NAD+, ADP, pyridoxal 5-phosphate), carbon isotope shifts of CO2 relative to the C1-carboxyl carbon of glycine varied from +14‰ to −7‰. The maximum effect of cofactors was observed for NAD+, the removal of which resulted in a strong 12C enrichment of the CO2 evolved. This indicates the possibility of isotope effects with both positive and negative signs in the GDC reaction. The measurement of δ13C in the leaves of the GDC-deficient barley (Hordeum vulgare L.) mutant (LaPr 87/30) plants indicated that photorespiratory carbon isotope fractionation, opposite in sign when compared to the carbon isotope effect during CO2 photoassimilation, takes place in vivo. Thus the key reaction of photorespiration catalyzed by GDC, together with the key reaction of CO2 fixation catalyzed by ribulose-1,5-bisphosphate carboxylase, both contribute to carbon isotope fractionation in photosynthesis.