Macromolecular synthesis of immune interferon (IFN-gamma) by the L12-R4 T cell lymphoma, stimulated by phorbol myristic acetate, was studied by using reversible inhibitors of protein synthesis, puromycin and cycloheximide, and an irreversible inhibitor of RNA synthesis, actinomycin D. Reversible inhibition of protein synthesis during the first 3 h of stimulation had no effect on IFN-gamma production. The same treatment, performed 4 h after stimulation and maintained for an additional 5 h, decreased significantly the capability of L12-R4 cells to produce IFN-gamma. When the inhibitors of protein synthesis were left in the cultures, a complete block of IFN-gamma production was observed. Irreversible inhibitors of RNA synthesis at the beginning of stimulation did block IFN-gamma production by L12-R4 cells, but the same treatment was ineffective if performed 6 h after stimulation. These data suggest that continued protein synthesis is needed for IFN-gamma production, whereas the RNA seems to be completely synthesized within 4 to 6 h of stimulation. The relationship between IFN-gamma production and cell cycle phases was studied with the aid of a reversible drug, aphidicolin, that arrests cells at the G1/S border. Phorbol myristic acetate stimulation of L12-R4 cells after aphidicolin removal induced comparable levels of IFN-gamma at each different point of stimulation, indicating that IFN-gamma production by stimulated cells is not related to a particular phase but is continuously inducible during the cell cycle.