Drought frequency and intensity are expected to increase in the Mediterranean as a consequence of global climate change. To understand how photosynthetic capacity responds to long-term water stress, we measured seasonal patterns of stomatal (S-L), mesophyll (MCL) and biochemical limitations (B-L) to net photosynthesis (A(max)) in three Quercus ilex (L.) populations from sites differing in annual rainfall. In the absence of water stress, stomatal conductance (g(s)), maximum carboxylation capacity (V-cmax), photosynthetic electron transport rate (J(max)) and A(max) were similar among populations. However, as leaf predawn water potential (Psi(l,pd)) declined, the population from the wettest site showed steeper declines in g(s), V-cmax, J(max) and A(max) than those from the drier sites. Consequently, S-L, MCL and B-L increased most steeply in response to decreasing Psi(l,pd) in the population from the wettest site. The higher sensitivity of A(max) to drought was primarily the result of stronger stomatal regulation of water loss. Among-population differences were not observed when g(s) was used instead of Psi(l,pd) as a drought stress indicator. Given that higher growth rates, stature and leaf area index were observed at the wettest site, we speculate that hydraulic architecture may explain the greater drought sensitivity of this population. Collectively, these results highlight the importance of considering among-population differences in photosynthetic responses to seasonal drought in large scale process-based models of forest ecosystem function