The presence of Foxp3(+) regulatory CD4(+) T cells in tumor lesions is considered one of the major causes of ineffective immune response in cancer. It is not clear whether intratumoral T(reg) cells represent T(reg) cells pre-existing in healthy mice, or arise from tumor-specific effector CD4(+) T cells and thus representing adaptive T(reg) cells. The generation of T(reg) population in tumors could be further complicated by recent evidence showing that both in humans and mice the peripheral population of T(reg) cells is heterogenous and consists of subsets which may differentially respond to tumor-derived antigens. We have studied T(reg) cells in cancer in experimental mice that express naturally selected, polyclonal repertoire of CD4(+) T cells and which preserve the heterogeneity of the T(reg) population. The majority of T(reg) cells present in healthy mice maintained a stable suppressor phenotype, expressed high level of Foxp3 and an exclusive set of TCRs not used by naive CD4(+) T cells. A small T(reg) subset, utilized TCRs shared with effector T cells and expressed a lower level of Foxp3. We show that response to tumor-derived antigens induced efficient clonal recruitment and expansion of antigen-specific effector and T(reg) cells. However, the population of T(reg) cells in tumors was dominated by cells expressing TCRs shared with effector CD4(+) T cells. In contrast, T(reg) cells expressing an exclusive set of TCRs, that dominate in healthy mice, accounted for only a small fraction of all T(reg) cells in tumor lesions. Our results suggest that the T(reg) repertoire in tumors is generated by conversion of effector CD4(+) T cells or expansion of a minor subset of T(reg) cells. In conclusion, successful cancer immunotherapy may depend on the ability to block upregulation of Foxp3 in effector CD4(+) T cells and/or selectively inhibiting the expansion of a minor T(reg) subset.