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Changes in climate, land management and fire regime have contributed to woody species expansion into grasslands and savannas worldwide. In the USA, Pinus ponderosa P. & C. Lawson and Juniperus virginiana L. are expanding into semiarid grasslands of Nebraska and other regions of the Great Plains. We examined P. ponderosa and J. virginiana seedling response to soil water content, one of the most important limiting factors in semiarid grasslands, to provide insight into their success in the region. Photosynthesis, stomatal conductance, maximum photochemical efficiency of PSII, maximum carboxylation velocity, maximum rate of electron transport, stomatal limitation to photosynthesis, water potential, root-to-shoot ratio, and needle nitrogen content were followed under gradual soil water depletion for 40 days. J. virginiana maintained lower Ls, higher A, gs, and initial Fv/Fm, and displayed a more gradual decline in Vcmax and Jmax with increasing water deficit compared to P. ponderosa. J. virginiana also invested more in roots relative to shoots compared to P. ponderosa. Fv/Fm showed high PSII resistance to dehydration in both species. Photoinhibition was observed at ~30% of field capacity. Soil water content was a better predictor of A and gs than Ψ, indicating that there are other growth factors controlling physiological processes under increased water stress. The two species followed different strategies to succeed in semiarid grasslands. P. ponderosa seedlings behaved like a drought-avoidant species with strong stomatal control, while J. virginiana was more of a drought-tolerant species, maintaining physiological activity at lower soil water content. Differences between the studied species and the ecological implications are discussed.