The presence of tumor-infiltrating lymphocytes (TIL) provides important evidence of anti-tumor immunity in vivo. However, TIL are usually not sufficient for inhibiting tumor growth. We explored the spatial and temporal aspects of clonal accumulation of TIL using RT-PCR/single-strand conformation polymorphism analysis. In CMS5 fibrosarcomas in BALB/c mice, accumulated T cell clones were specific in that dominant TIL were identical between distant tumors. Moreover, dominant TIL in the first tumor appeared consistently in the second tumor inoculated after formation of the first tumor. These results suggest that TIL show a certain level of specific tumor surveillance. When we characterized CD4(+) and CD8(+) TIL separately, CD8(+) TIL were highly concentrated and persistently localized at the tumor site, while most CD4(+) TIL clones were less concentrated and less persistent. A functional analysis showed that TIL had a certain degree of anti-tumor activity when CD4(+) and CD8(+) TIL were co-transferred. Co-transfer of CD4(+) and CD8(+) TIL exhibited equivalent anti-tumor activity, irrespective of tumor stage. However, the numbers of TIL did not increase after the early phase of tumor progression. These data suggest that TIL are specific to the tumor and potentially retain anti-tumor activity, although their accumulation in mice is impaired.