Tumor infiltration by lymphocytes has been linked to improved clinical outcome in children with neuroblastoma (NB) but T-cell activation has never been demonstrated to occur within the NB microenvironment. Here we show that tumor-associated lymphocytes (TALs) obtained from lesions representing all genetic subsets of NB and autologous peripheral blood lymphocytes (PBLs) analyzed on the day of tumor excision differed in composition, phenotype and functional characteristics. The NB microenvironment appeared to promote the accumulation of CD3+CD8+ T cells and contained a larger proportion of T cells expressing the interleukin-2 receptor α chain (CD25) and manifesting an effector memory (CCR7−:CD45RA−:) phenotype. Accordingly, the stimulation of PBLs with autologous tumor cells in short-term cultures increased the proportion of effector memory T cells, upregulated CD25, stimulated the expression of the TH1 cytokines interferon γ and tumor necrosis factor α, and reduced the expression of transforming growth factor β. In situ proliferation as well as a characteristic pattern of T-cell receptor aggregation at the contact sites with malignant cells was revealed by the immunohistochemical staining of TALs in primary tumors, indicating that the NB milieu is compatible with the activation of the immune system. Our results are compatible with the hypothesis that CD8+ T cells are specifically activated within the NB microenvironment, which appears to be permissive for effector memory responses.