Abstract. Lake Chala (3°19′ S, 37°42′ E) is a steep-sided crater lake situated in equatorial East Africa, a tropical semiarid area with a bimodal rainfall pattern. Plants in this region are exposed to a prolonged dry season, and we investigated if (1) these plants show spatial variability and temporal shifts in their water source use; (2) seasonal differences in the isotopic composition of precipitation are reflected in xylem water; and (3) plant family, growth form, leaf phenology, habitat and season influence the xylem-to-leaf water deuterium enrichment. In this study, the δ2H and δ18O of precipitation, lake water, groundwater, plant xylem water and plant leaf water were measured across different plant species, seasons and plant habitats in the vicinity of Lake Chala. We found that plants rely mostly on water from the short rains falling from October to December (northeastern monsoon), as these recharge the soil after the long dry season. This plant-available, static water pool is only slightly replenished by the long rains falling from February to May (southeastern monsoon), in agreement with the two water worlds hypothesis, according to which plants rely on a static water pool while a mobile water pool recharges the groundwater. Spatial variability in water resource use exists in the study region, with plants at the lakeshore relying on a water source admixed with lake water. Leaf phenology does not affect water resource use. According to our results, plant species and their associated leaf phenology are the primary factors influencing the enrichment in deuterium from xylem water to leaf water (εl/x), with deciduous species giving the highest enrichment, while growth form and season have negligible effects. Our observations have important implications for the interpretation of δ2H of plant leaf wax n-alkanes (δ2Hwax) from paleohydrological records in tropical East Africa, given that the temporal variability in the isotopic composition of precipitation is not reflected in xylem water and that leaf water deuterium enrichment is a key factor in shaping δ2Hwax. The large interspecies variability in xylem–leaf enrichment (24 ± 28 ‰) is potentially troublesome, taking into account the likelihood of changes in species assemblage with climate shifts.