The responses of B16 melanoma in C57BL mice to cytotoxic agents, cyclophosphamide, doxorubicin, and dactinomycin, were studied by different methods both in vivo and in vitro. Either treated tumors were allowed to grow in vivo or cells were isolated and cultured in vitro afterwards. The tumor cells were also treated in vitro and assayed in vitro. For cyclophosphamide, a fairly good correlation was found among the dose responses measured: the tumor growth delay, incidence of giant cells, decrease in mitotic index, clonogenic cell survival of tumor cells treated in vivo and the survival of cells treated in vitro. For doxorubicin and dactinomycin, on the other hand, the tumor growth delay was not marked, the appearance of giant cells and decrease in mitotic index were minimal, and almost no decrease was found in the clonogenic cell survival for tumor treated in vivo, although the cells responded well to in vitro exposure. The results indicate substantial differences in the expression of damage according to the conditions of tumor cell growth and assay after treatment with different agents.