We evaluated diurnal and seasonal patterns of carbon isotope composition of leaf dark-respired CO2 (δ13Cl) in the C3 perennial shrub velvet mesquite (Prosopis velutina) across flood plain and upland savanna ecosystems in the south-western USA. δ13Cl 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 δ13Cl was strongly influenced by seasonal and habitat-related differences in soil water availability and leaf surface vapour pressure deficit. δ13Cl and the cumulative flux-weighted δ13C value of photosynthates were positively correlated, suggesting that progressive 13C enrichment of the CO2 evolved by darkened leaves during the daytime mainly resulted from short-term changes in photosynthetic 13C discrimination and associated shifts in the δ13C signature of primary respiratory substrates. The 13C enrichment of dark-respired CO2 relative to photosynthates across habitats and seasons was 4 to 6‰ at the end of the daytime period (1800 h), but progressively declined to 0‰ by pre-dawn (0300 h). The origin of night-time and daytime variations in δ13Cl is discussed in terms of the carbon source(s) feeding respiration and the drought-induced changes in carbon metabolism.