Accumulation of T cells at the tumor is essential in cancer immunotherapy based on adoptive transfer of tumor-specific T cells. To gain further insight into the accumulation process and to evaluate the effect of using different routes of cell transfer, we investigated the accumulation of ovalbumin-specific CD8+ T cells (OT-I) injected either intravenously (IV) or intraperitoneally (IP) into mice carrying a subcutaneous tumor of the ovalbumin-expressing melanoma cell line B16-OVA. Maximal accumulation of the adoptively transferred cells in tumor tissue was observed 5 days after injection, irrespective of the injection route. The route of injection affected neither the total number of adoptively transferred cells found in tumor tissue nor the kinetics of this accumulation. In the spleen, however, the accumulation of adoptively transferred cells was clearly dependent on the injection route. IP injections resulted in a large number of adoptively transferred cells in the spleen on all days analyzed. In comparison, IV injection resulted in significantly fewer adoptively transferred cells in the spleen, and this number decreased over time. The route of injection affected neither the activation status of the adoptively transferred T cells that accumulated at the tumor site, nor the ability of these cells to control tumor growth. Two cell populations, SIINFEKL-tetramerLow(TetLow)CD69+CD25+ and TetHighCD69−CD25−, were present in tumor samples, whereas only TetHighCD69−CD25− cells accumulated in the spleen. In tumors, IV injection resulted in a higher fraction of adoptively transferred cells with an activated phenotype (TetLowCD69+CD25+) compared with IP injection.