The evolution of vacancy-type crystalline defects across the transition from anatase to rutile structure of titanium dioxide (TiO2) nanoparticles during high-temperature annealing in oxygen and argon is studied by using positron lifetime and coincidence Doppler broadening spectroscopic measurements. The TiO2 nanoparticles were synthesized through a simple sol-gel chemical route. The changes in the crystalline phase and lattice parameters of the nanoparticles upon thermal treatment were investigated by X-ray diffraction and high-resolution transmission electron microscopy, and the results were correlated with those of photoluminescence spectroscopy and positron annihilation measurements. The structural defects, mostly 3D vacancy clusters, in the nanoparticles were found to decrease in concentration during the annealing in O2 rather than in Ar at elevated temperatures. In the case of annealing in Ar, the vacancy-type defects persisted even at the highest annealing temperature of 900 °C used in the experiment and the transition was, as a result, found to be delayed and partial. The annihilation of positrons at the nanocrystalline grain interfaces also contributed to the long positron lifetime component, but variations due to annealing were clearly visible as it also contained the contributions from annihilation within the vacancy clusters. The role of the vacancy-type defects on the magnetic property of the TiO2 nanoparticles is also investigated. © 2013 American Chemical Society.