Water availability is the most important factor for the vitality of forest ecosystems, especially in dry environments. The Mediterranean region is one of the hotspots of future climate change; therefore, data on the water cycle are urgently needed. We measured oxygen isotope compositions in creek water, precipitation, stem water, needle water, and tree-ring cellulose over one growing season to establish the relationship between isotope compositions in different compartments along a fractionation pathway. We analyzed plant material from pine trees (Pinus nigra J.F. Arn subsp. laricio (Poiret) Maire var. Corsicana Hyl. and Pinus pinaster Aiton) at five locations along an elevation gradient from sea level to 1600 m asl. We traced back the oxygen isotope composition from source to sink in tree-ring cellulose in order to identify the water sources used by the trees, and to quantify the extent of isotope fractionation processes. Our results showed that the trees used different water sources over the course of the growing season, ranging from winter snow meltwater to summer precipitation at higher sites and deep soil water reservoirs at coastal sites. Needle water enrichment was higher at higher elevation sites than at coastal locations, highlighting the importance of site-specific climate conditions on the isotopic composition values in tree material. Water availability seems to be most restricted at the highest site, making these trees most vulnerable to climate change.