Plant transpiration is a major component of water fluxes in the critical zone, which needs to be better characterized to improve our ability to understand and model the hydrological cycle. In water-limited ecosystems such as those encountered on karst environments, climate-induced changes in transpiration are expected to be strongly influenced by the ability of the vegetation cover to resist or adapt to drought. However, because of the high heterogeneity of karst environments, the amount of water available for trees can change within a stand, which may lead to significant differences in drought vulnerability resistance between trees of the same species. So far it is not known if soil and subsoil environment influence the magnitude of deep water extraction, at the intra-specific scale. Here, we investigate the variability in deep water extraction for six individual Quercus ilex trees growing on a karst substrate in a Mediterranean forest. We combined three approaches: (i) electrical resistivity tomography to determine the variability of soil/subsoil characteristics, (ii) isotope tracing to determine the origin of water transpired by plants, and (iii) predawn and midday leaf water potential (Ψ) to assess the trees’ water stress and transpiration regulation. Along the summer season, deep water extraction increased with drought intensity. Deep water use varies between individuals and according to drought intensity. At moderate water stress levels, we found no significant relationship between the origin of xylem water and soil/subsoil characteristics or individual stress level. However, at the peak of the drought (average predawn Ψ < −2 MPa), individuals that had the least total available water in soil/subsoil (0–2 m) relied more on deep water and were also subject to less water stress. These results suggest that trees with less favorable soil/subsoil conditions (i.e. low water retention capacity) in the near surface (0–2 m) adapt their root systems to exploit deep water reserves more intensively so as to enhance their drought tolerance, while trees with more favorable surface conditions exhibit greater water stress and may be more vulnerable to extreme droughts because of a lower root development in deeper horizons.