The in vitro cytotoxic effect of spleen cells of mice immunized by tumour allografts was studied by measuring target cell inactivation as a function of release of radioactive label (51Cr) or loss of cloning efficiency. When sensitized lymphoid cells were incubated with target cells at a ratio of 100:1, up to 90 per cent of the incorporated label was released within 6–9 hours, while the number of clone-forming cells was reduced by up to 99 per cent in the same time period. Isoantiserum from the graft recipients, as well as its 19S and 7S fractions, protected target cells against the toxic effect of the spleen cells, but a lipoprotein antigen isolated from the tumour cells failed to inhibit the cytotoxic reaction. Target cell lysis as measured by specific release of 51Cr was partially inhibited by actinomycin-D and by cycloheximide at concentrations which effectively blocked DNA-dependent RNA and protein synthesis.