Acquisition of full T-cell effector function and memory differentiation requires appropriate costimulatory signals, including ligation of the costimulatory molecule OX40 (TNFRSF4, CD134). Tumors often grow despite the presence of tumor-specific T cells and establish an environment with weak costimulation and immune suppression. Administration of OX40 agonists has been shown to significantly increase the survival of tumor-bearing mice and was dependent on the presence of both CD4 and CD8 T cells during tumor-specific priming. To understand how OX40 agonists work in mice with established tumors, we developed a model to study changes in immune cell populations within the tumor environment. We show here that systemic administration of OX40 agonist antibodies increased the proportion of CD8 T cells at the tumor site in three different tumor models. The function of the CD8 T cells at the tumor site was also increased by administration of OX40 agonist antibody, and we observed an increase in the proportion of antigen-specific CD8 T cells within the tumor. Despite decreases in the proportion of T regulatory cells at the tumor site, T regulatory cell function in the spleen was unaffected by OX40 agonist antibody therapy. Interestingly, administration of OX40 agonist antibody caused significant changes in the tumor stroma, including decreased macrophages, myeloid-derived suppressor cells, and decreased expression of transforming growth factor-β. Thus, therapies targeting OX40 dramatically changed the tumor environment by enhancing the infiltration and function of CD8 T cells combined with diminished suppressive influences within the tumor. [Cancer Res 2008;68(13):5206–15]