The first broad species survey of diurnal variation in carbon (C) isotope signatures of leaf dark-respired CO(2) (delta(13)C(res)) is presented here and functional differences and diurnal dynamics are linked to fractionation in different respiratory pathways, based on (13)C-labelling experiments. delta(13)C(res) was analysed with a rapid in-tube incubation technique in 16 species. A large diurnal increase in delta(13)C(res) (4-8 per thousand) occurred in evergreen, slow-growing and aromatic species and correlated significantly with cumulative photosynthesis, whereas no variation occurred in herbaceous, fast-growing plants or temperate trees. The diurnal increase in delta(13)C(res) declined almost proportionally to reductions in cumulative light and was reduced in growing compared with mature leaves. Pyruvate positional labelling provided direct evidence that functional groups differ in C allocation between respiratory pathways owing to different metabolic demands for growth, maintenance and secondary metabolism. Diurnal increase in C flux through pyruvate dehydrogenase (for investment in, for example, isoprene or aromatic compounds) combined with consistently low Krebs cycle activity resulted in pronounced increase in delta(13)C(res) in evergreen and aromatic species. By contrast, fast growing herbs with high respiratory demand exhibited no diurnal changes since C was fully respired. Hence, diurnal delta(13)C(res) pattern may provide information for C allocation in plants.