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The root-zone moisture replenishment mechanisms are key unknowns required to understand soil hydrological processes and water sources used by plants. Temporal patterns of root-zone moisture replenishment reflect wetting events that contribute to plant growth and survival and to catchment water yield. In this study, stable oxygen and hydrogen isotopes of twigs and throughfall were continuously monitored to characterize the seasonal variations of the root-zone moisture replenishment in a native vegetated catchment under Mediterranean climate in South Australia. The two studied hillslopes (the north-facing slope [NFS] and the south-facing slope [SFS]) had different environmental conditions with opposite aspects. The twig and throughfall samples were collected every similar to 20 days over 1 year on both hillslopes. The root-zone moisture replenishment, defined as percentage of newly replenished root-zone moisture as a complement to antecedent moisture for plant use, calculated by an isotope balance model, was about zero (+/- 25% for the NFS and +/- 15% for the SFS) at the end of the wet season (October), increased to almost 100% (+/- 26% for the NFS and +/- 29% for the SFS) after the dry season (April and May), then decreased close to zero (+/- 24% for the NFS and +/- 28% for the SFS) in the middle of the following wet season (August). This seasonal pattern of root-zone moisture replenishment suggests that the very first rainfall events of the wet season were significant for soil moisture replenishment and supported the plants over wet and subsequent dry seasons, and that NFS completed replenishment over a longer time than SFS in the wet season and depleted the root zone moisture quicker in the dry season. The stable oxygen isotope composition of the intraevent samples and twigs further confirms that rain water in the late wet season contributed little to root-zone moisture. This study highlights the significant role of the very first rain events in the early wet season for ecosystem and provides insights to understanding ecohydrological separation, catchment water yield, and vegetation response to climate changes.