Mitogen-induced interferon-gamma (IFN-gamma) gene expression was analyzed in human tonsil cells by titration of IFN-gamma activity and by quantitation of IFN-gamma mRNA. Expression of the IFN-gamma gene can be superinduced extensively by two distinct methods: exposure to various inhibitors of translation, or to low doses of gamma-irradiation. gamma-Irradiated cells produce, after exposure to cycloheximide, up to 12-fold greater amounts of IFN-gamma activity. Within as little as 4 h after the addition of translation inhibitors, IFN-gamma mRNA levels rise 3- to 5-fold. Superinduction acts to increase the size of the wave of IFN-gamma mRNA. Primary transcription of the IFN-gamma gene does not increase in cells superinduced by cycloheximide, nor can superinduction be explained by stabilization of IFN-gamma mRNA sequences. These findings show that, during normal induction, a labile protein acts post-transcriptionally to repress the accumulation of mature IFN-gamma mRNA sequences. The superinductive effects of cycloheximide and gamma-irradiation on levels of IFN-gamma are additive, suggesting that they affect different aspects of IFN-gamma gene expression. Superinduction by gamma-irradiation also has a post-transcriptional basis and is consistent with the possibility that expression of the IFN-gamma gene is normally controlled by the action of suppressor T cells. Even though the genes for human IFN-gamma and for interleukin-2 are both superinducible, a striking difference in the regulation of expression of these lymphokine genes is observed. Superinduction of IFN-gamma mRNA is not due to superinduction of interleukin-2.