Cytotoxic T lymphocytes (CTL) for MM tumor cells were generated only when L-MM tumor hybrid (L-FM3A#2)-primed spleen cells were stimulated in vitro with a mixture of mitomycin C-treated parental FM3A/R tumor cells and L cells, but not when they were stimulated with FM3A/R or L cells alone. Killer-helper activity of L cells was replaced by a beta-interferon (IFN) preparation (L-cell IFN). Anti-(alpha + beta)-IFN antibodies completely inhibited the killer-helper activity of L-cell IFN. Several lines of evidence suggested that the killer-helper effect of L-cell IFN resulted from an action at an early step in the generation of anti-MM CTL, as follows. The killer-helper effect of L-cell IFN diminished as the addition was delayed during culture for 5 days. The presence of L-cell IFN during the first 24 hr was enough to generate a maximal CTL response. Anti-(alpha + beta)-IFN antibodies showed no inhibitory activity on the generation of anti-MM CTL when they were added to the culture on day 2 or 4. It was also demonstrated that the killer-helper effect of L-cell IFN resulted from an effect on primed spleen cells, but not from an effect on stimulator cells. Furthermore, it was observed that IFN acted on nylon wool-adherent cells in the generation of anti-MM CTL. Plastic-adherent cells preincubated with L-cell IFN also enhanced the generation of anti-MM CTL when they were added to a culture where primed spleen cells were stimulated with FM3A/R tumor cells. These results suggest that activation of macrophages by L-cell IFN at the initiation step is essential for the generation of anti-MM CTL.