We have carried out positron-annihilation spectroscopy to characterize the spatial distribution and the nature of vacancy defects in insulating as-received as well as in reduced SrTiO 3 substrates exhibiting high-mobility conduction. The substrates were reduced either by ion etching the substrate surfaces or by doping with vacancies during thin-film deposition at low pressure and high temperature. We show that Ti vacancies are native defects homogeneously distributed in as-received substrates. In contrast, the dominant vacancy defects are the same both in ion etched crystals and substrates reduced during the film growth, and they consist of nonhomogeneous distributions of cation-oxygen vacancy complexes. Their spatial extension is tuned from a few microns in ion-etched samples to the whole substrate in specimens reduced during film deposition. Our results shed light on the transport mechanisms of conductive SrTiO 3 crystals and on strategies for defectengineered oxide quantum wells, wires, and dots.