Recent advances in understanding the metabolic origin and the temporal dynamics in
δ13C of dark‐respired CO
2 (
δ13C
res) have led to an increasing awareness of the importance of plant isotopic fractionation in respiratory processes. Pronounced dynamics in
δ13C
res have been observed in a number of species and three main hypotheses have been proposed: first, diurnal changes in
δ13C of respiratory substrates; second, post‐photosynthetic discrimination in respiratory pathways; and third, dynamic decarboxylation of enriched carbon pools during the post‐illumination respiration period. Since different functional groups exhibit distinct diurnal patterns in
δ13C
res (ranging from 0 to 10‰ diurnal increase), we explored these hypotheses for different ecotypes and environmental (i.e. growth light) conditions. Mass balance calculations revealed that the effect of respiratory substrates on diurnal changes in
δ13C
res was negligible in all investigated species. Further, rapid post‐illumination changes in
δ13C
res (30 min), which increased from 2.6‰ to 5‰ over the course of the day, were examined by positional
13C‐labelling to quantify changes in pyruvate dehydrogenase (PDH) and Krebs cycle (KC) activity. We investigated the origin of these dynamics with Rayleigh mass balance calculations based on theoretical assumptions on fractionation processes. Neither the estimated changes of PDH and KC, nor decarboxylation of a malate pool entirely explained the observed pattern in
δ13C
res. However, a Rayleigh fractionation of
12C‐discriminating enzymes and/or a rapid decline in the decarboxylation rate of an enriched substrate pool may explain the post‐illumination peak in
δ13C
res. These results are highly relevant since
δ13C
res is used in large‐scale carbon cycle studies. Copyright © 2009 John Wiley & Sons, Ltd.
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