The C isotope composition of leaf dark-respired CO2 (delta C-13(l)) integrates short-term metabolic responses to environmental change and is potentially recorded in the isotopic signature of ecosystem-level respiration. Species differences in photosynthetic pathway, resource acquisition and allocation patterns, and associated isotopic fractionations at metabolic branch points can influence delta C-13(l), and differences are likely to be modified by seasonal variation in drought intensity. We measured delta C-13(l) in two deep-rooted C-3 trees (Prosopis velutina and Celtis reticulata), and two relatively shallow-rooted perennial herbs (a C-3 dicot Viguiera dentata and a C-4 grass Sporobolus wrightii) in a floodplain savanna ecosystem in southeastern Arizona, USA during the dry pre-monsoon and wet monsoon seasons. delta C-13(l) decreased during the nighttime and reached minimum values at pre-dawn in all species. The magnitude of nocturnal shift in delta C-13(l) differed among species and between pre-monsoon and monsoon seasons. During the pre-monsoon season, the magnitude of the nocturnal shift in delta C-13(l) in the deep-rooted C-3 trees P. velutina (2.8 +/- A 0.4aEuro degrees) and C. reticulata (2.9 +/- A 0.2aEuro degrees) was greater than in the C-3 herb V. dentata (1.8 +/- A 0.4aEuro degrees) and C-4 grass S. wrightii (2.2 +/- A 0.4aEuro degrees). The nocturnal shift in delta C-13(l) in V. dentata and S. wrightii increased to 3.2 +/- A 0.1aEuro degrees and 4.6 +/- A 0.6aEuro degrees, respectively, during the monsoon season, but in C-3 trees did not change significantly from pre-monsoon values. Cumulative daytime net CO2 uptake was positively correlated with the magnitude of the nocturnal decline in delta C-13(l) across all species, suggesting that nocturnal delta C-13(l) may be controlled by C-13/C-12 fractionations associated with C substrate availability and C metabolite partitioning. Nocturnal patterns of delta C-13(l) in dominant plant species in the semiarid savanna apparently have predictable responses to seasonal changes in water availability, which is important for interpreting and modeling the C isotope signature of ecosystem-respired CO2.