This study attempts to characterize human melanoma-associated tumor antigens that are recognized by autologous T-lymphocyte clones. Peripheral blood lymphocytes of a melanoma patient (AV) were activated in an autologous-mixed-lymphocyte tumor culture (AMLTC) and responding cells were cloned using irradiated autologous melanoma cells (AV-Mel) as antigen source, EBV-transformed B-lymphoblasts as feeder cells and recombinant Interleukin 2. T-cell clones were isolated which proliferated in response to autologous tumor cells, but not to autologous B-lymphoblasts (AV-B), and which were cytolytic for AV-Mel cells. In an attempt to identify the antigens recognized, an in vitro test system was used, in which 3H-Thymidine (3H-TdR) incorporation by T lymphocytes was measured in the presence of protein from AV-Mel cells presented by irradiated autologous accessory cells. Antigen-bearing particles of AV-Mel or AV-B cells were prepared by spotting cell lysates onto nitrocellulose (NC) followed by dissolution with DMSO and precipitation with an aqueous buffer. T cells sensitized against autologous melanoma cells were specifically stimulated by NC-bound AV-Mel protein. Stimulation required the presence of AV-B accessory cells, indicating that B-lymphoblasts are able to present tumor antigens. This approach facilitates screening of polypeptide fractions of AV-Mel cells separated by polyacrylamide gel electrophoresis followed by Western blotting for their capacity to stimulate antigen-dependent T-cells. CD4+ and CD8+ tumor-specific clones appear to recognize different antigens on the tumor cell: proliferation of an antigen-dependent CD8+ clone was stimulated by 240- and 24-kDa protein fractions, while proliferation of 2 antigen-dependent CD4+ clones was observed either with an 84- or with 140- and 55-kDa fractions. Molecular definition of the different antigens of tumor cells recognized by autologous T cells may be a prerequisite in attempts to manipulate T-cell-mediated anti-tumor responses in a controlled way.