Au nanoparticles supported on reducible metal oxide surfaces are known to be active catalysts for a number of reactions including CO oxidation and hydrogen production. The exact choice of a metal oxide support has been shown to have a marked impact on activity, suggesting that interactions between Au and the support play a key role in catalysis. For TiO2, a model substrate for Au catalysis, it had been thought that bridging oxygen vacancies are involved in binding Au atoms to the (110) surface based on indirect evidence. However, a recent scanning transmission electron microscopy study of single Pt atoms on TiO2(110) suggests that subsurface vacancies are more important. To clarify the role of bridging or subsurface vacancies we employ scanning tunneling microscopy to determine the bonding site of single Au atoms on TiO2(110). Using in situ deposition as well as a manipulation method, we provide definitive evidence that the bonding site is atop surface oxygen vacancies.