Accurate estimates of the d13C value of CO2 respired from roots (d13CR_root) and leaves (d13CR_leaf) are important for tracing and understanding changes in C fluxes at the ecosystem scale. Yet the mechanisms underlying temporal variation in these isotopic signals are not fully resolved. We measured d13CR_leaf, d13CR_root, and the d13C values and concentrations of glucose and sucrose in leaves and roots in the C4 grass Sporobolus wrightii and the C3 tree Prosopis velutina in a savanna ecosystem in southeastern Arizona, USA. Night-time variation in d13CR_leaf of up to 4.6 +/- 0.6 parts per thousand in S. wrightii and 3.0 +/- 0.6 parts per thousand in P. velutina were correlated with shifts in leaf sucrose concentration, but not with changes in d13C values of these respiratory substrates. Strong positive correlations between d13CR_root and root glucose d13C values in P. velutina suggest large diel changes in d13CR_root (were up to 3.9 parts per thousand) influenced by short-term changes in d13C of leaf-derived phloem C. No diel variation in d13CR_root was observed in S. wrightii. Our findings show that short-term changes in d13CR_leaf and d13CR_root were both related to substrate isotope composition and concentration. Changes in substrate limitation or demand for biosynthesis may largely control short-term variation in the d13C of respired CO2 in these species.