We have reported that medium containing recombinant human IL-1 (rIL-1), rIL-2, rIL-4, and rIL-6 (MB-1,2,4,6 medium) efficiently expanded autologous tumor specific CTLs in vitro. For further examination of the CTLs cultured in MB-1,2,4,6, the therapeutic activity on tumor growth inhibition in vivo and established CTL clones were studied. In vivo therapy with the CTLs in combination with rIL-2 was highly effective. To investigate CTL clones, 19 CD8+ T cell clones were obtained by limiting dilution method, each clone retained autologous-melanoma-specific, HLA-class I-restricted cytolytic activity. Four T cell clones were analyzed in detail. These T cell clones displayed a CD3+, CD4-, CD8+, CD11b-, CD16-, CD56-, CD45RA-, TcR alpha/beta + phenotype and monoclonal antibodies to HLA class I, CD3, and CD8 antigens inhibited their cytolytic activity. Moreover, these CTL clones recognize one of common melanoma antigens associated with HLA-B (B49). Analysis of the effect of different cytokines on the proliferation and cytotoxicity of cloned CTL revealed the highest growth rate in MB-1,2,4,6 but no dependence on particular cytokine combinations for autologous tumor-specific cytolytic activity. These results suggest (1) the usefulness of MB-1,2,4,6 medium in expanding autologous tumor-specific CTLs, which can be used in adoptive immunotherapy, (2) HLA-B molecules can present one of common melanoma antigens, and (3) the independence of CTLs from any cytokine combination once they become target-specific.