Scanning tunneling microscopy (STM) and spectroscopy have been used to investigate the structure of rutile TiO2(100)1 X 3. The combination of these two techniques allows us to identify individual oxygen vacancies on the 1 X 3 reconstructed surface. These vacancies are found to occupy the topmost layer of the surface and to form one-dimensional arrays in the [001] crystallographic direction, with an intervacancy separation of 2.96 angstrom and a typical length of approximately 500 angstrom. The STM data are consistent with a microfacet structural model proposed on the basis of grazing incidence x-ray diffraction data, modified to include the presence of 0 vacancies in the top layer.