Young secondary tropical forests occupy a larger area than mature forests nowadays but our understanding of their ecohydrological functioning, particularly with respect to tree water uptake, remains poor. Deep soil water uptake may make mature forests resilient to periods of water stress, but little is known in this regard for young forests with possibly less extensive root networks. We, therefore, studied sap flow dynamics for one year in two 50 m x 50 m forest plots: a young secondary forest (YSF, 5–7 years) and a semi-mature forest (SMF; 20 years) in montane eastern Madagascar. Temporal variations in the depth of water uptake were inferred from the stable isotope compositions of soil- and xylem water. Transpiration rates were low for both forest sites (265 and 462 mm y−1 for the YSF and SMF, respectively). Vapour pressure deficit and global radiation explained most of the variation in transpiration rates at both sites. There was little evidence of transpiration limitation by soil water, despite an extended dry season. Trees in the YSF extracted water mostly from the intermediate soil depth (30–70 cm) during the dry season. In the SMF, the depth of uptake increased as the dry season progressed for some species (Abrahamia, Brachylaena and Cryptocaria), but not for others (Ocotea and Eugenia). Although the transpiration rates are low for both forests, they are comparable to results reported for other tropical montane sites after normalising for net energy input and leaf area. Estimated evapotranspiration totals (including interception loss, understorey and litter evaporation) were 679 mm and 1063 mm y−1 for the YSF and SMF, respectively (42% and 61% of precipitation, respectively). These results suggest that the stage of forest regrowth affects water uptake, and thus the water balance during forest succession.