Water-use efficiency (WUE), thought to be a relevant trait for productivity and adaptation to water-limited environments, was estimated for three different ecosystems on the Mediterranean island of Pianosa: Mediterranean macchia (S-MM), transition (S-TR) and abandoned agricultural (S-AA) ecosystems, representing a successional series. Three independent approaches were used to study WUE: eddy covariance measurements, C isotope composition of ecosystem respired CO2, and C isotope discrimination (Delta) of leaf material (dry matter and soluble sugars). Seasonal variations in C-water relations and energy fluxes, compared in S-MM and in S-AA, were primarily dependent on the specific composition of each plant community. WUE of gross primary productivity was higher in S-MM than in S-AA at the beginning of the dry season. Both structural and fast-turnover leaf material were, on average, more enriched in C-13 in S-MM than S-AA, indicating relatively higher stomatal control and WUE for the long-lived macchia species. This pattern corresponded to C-13-enriched respired CO2 in S-MM compared to the other ecosystems. Conversely, most of the annual herbaceous S-AA species (terophytes) showed a drought-escaping strategy, with relatively high stomatal conductance and low WUE. An ecosystem-integrated Delta value was weighted for each ecosystem on the abundance of different life forms, classified according to Raunkiar's system. Agreement was found between ecosystem WUE calculated using eddy covariance and those estimated using integrated Delta approaches. Comparing the isotopic methods, Delta of leaf soluble sugars provided the most reliable proxy for short-term changes in photosynthetic discrimination and associated shifts in integrated canopy-level WUE along the successional series.