Treatment of metastatic melanoma patients with an autologous vaccine modified by the hapten, dinitrophenyl (DNP), produces a striking immunological effect: the induction of clinically evident inflammatory responses in metastatic tumors. Histological examination shows these tumors to be infiltrated with T lymphocytes. We studied the expression of activation markers on those cells and compared them with matched peripheral blood lymphocytes (PBL) and with lymphocytes extracted from metastases before treatment with DNP-conjugated vaccine. The median fraction of cells that were T cells in post-vaccine tumors was 41%, as compared with 9% in pre-treatment tumors, and those T cells were predominantly CD8+ (mean CD8/CD4 ratio = 5.0). A high proportion of both pre- and post-treatment infiltrating T cells expressed HLA-DR (mean +/- SE = 48% +/- 4%), CD69 (56% +/- 7%), and ganglioside GD3 (68% +/- 5%). This distinguished them from matched PBL in which expression of those markers was significantly lower (HLA-DR = 10% +/- 2%; CD69 = 2% +/- 0.4%; GD3 = 49% +/- 4%). These changes were not accompanied by increased cell-surface expression of interleukin-2 (IL-2) receptors, either CD25 or p75, which were expressed by 1%-2% and 12% of tumor-infiltrating lymphocytes (TIL), respectively. The pattern of activation marker expression that we identified appears to be characteristic of tissue T cells with the memory phenotype. The low expression of IL-2 receptors could indicate functional impairment of TIL in situ, perhaps because of inhibitory molecules produced by melanoma cells.