The (13)C isotopic signature (delta(13)C) of CO(2) respired from plants is widely used to assess carbon fluxes and ecosystem functioning. There is, however, a lack of knowledge of the metabolic basis of the delta(13)C value of respired CO(2). To elucidate the physiological mechanisms driving (12)C/(13)C fractionation during respiration, the delta(13)C of respired CO(2) from dark-acclimated leaves during the night, from darkened leaves during the light period, and from stems and roots of Ricinus communis was analysed. The delta(13)C of potential respiratory substrates, the respiratory quotient and the activities of phosphoenolpyruvatecarboxylase (PEPc) and key respiratory enzymes were also measured. It is shown here that the CO(2) evolved from darkened light-acclimated leaves during the light period is (13)C-enriched, and that this correlates with malate accumulation in the light and rapid malate decarboxylation just after the onset of darkness. Whilst CO(2) evolved from leaves was generally (13)C-enriched (but to a lesser extent during the night), CO(2) evolved from stems and roots was depleted compared with the putative respiratory substrates; the difference was mainly caused by intensive PEPc-catalysed CO(2) refixation in stems and roots. These results provide a physiological explanation for short-term variations of delta(13)C in CO(2), illustrating the effects of variations of metabolic fluxes through different biochemical pathways.