We evaluated diurnal and seasonal patterns of carbon isotope composition of leaf dark-respired CO2 (delta C-13(l)) in the C-3 perennial shrub velvet mesquite (Prosopis velutina) across flood plain and upland savanna ecosystems in the south-western USA. delta C-13(l) of darkened leaves increased to maximum values late during daytime periods and declined gradually over night-time periods to minimum values at pre-dawn. The magnitude of the diurnal shift in delta C-13(l) was strongly influenced by seasonal and habitat-related differences in soil water availability and leaf surface vapour pressure deficit. delta C-13(l) and the cumulative flux-weighted delta C-13 value of photosynthates were positively correlated, suggesting that progressive C-13 enrichment of the CO2 evolved by darkened leaves during the daytime mainly resulted from short-term changes in photosynthetic C-13 discrimination and associated shifts in the delta C-13 signature of primary respiratory substrates. The C-13 enrichment of dark-respired CO2 relative to photosynthates across habitats and seasons was 4 to 6 parts per thousand at the end of the daytime period (1800 h), but progressively declined to 0 parts per thousand by pre-dawn (0300 h). The origin of night-time and daytime variations in delta C-13(l) is discussed in terms of the carbon source(s) feeding respiration and the drought-induced changes in carbon metabolism.