Summary• As described in the two-layer hypothesis, woody plants are often assumed to use deep soils to avoid competition with grasses. Yet the direct measurements of root activity needed to test this hypothesis are rare.• Here, we injected deuterated water into four soil depths, at four times of year, to measure the vertical and horizontal location of water uptake by trees and grasses in a mesic savanna in Kruger National Park, South Africa.• Trees absorbed 24, 59, 14 and 4% of tracer from the 5, 20, 50, and 120 cm depths, respectively, while grasses absorbed 61, 29, 9 and 0.3% of tracer from the same depths. Only 44% of root mass was in the top 20 cm. Trees absorbed tracer under and beyond their crowns, while 98% of tracer absorbed by grasses came from directly under the stem.• Trees and grasses partitioned soil resources (20 vs 5 cm), but this partitioning did not reflect, as suggested by the two-layer hypothesis, the ability of trees to access deep soil water that was unavailable to grasses. Because root mass was a poor indicator of root activity, our results highlight the importance of precise root activity measurements.