Inhibitors of RNA synthesis such as actinomycin D, 2-mercapto-1-(beta-4-pyridylethyl)benzimidazole, and cordycepin progressively inhibit the initiation of protein synthesis in intact nucleated mammalian cells independent of their effect on mRNA synthesis. The mechanism of this effect is unknown. The activity of cell-free lysates is not directly affected by these inhibitors, suggesting that their effect is indirect and requires an intact cell. However, lysates prepared from L-cells or CHO cells treated with the inhibitors do exhibit a decrease in initiation activity corresponding in magnitude to the effect seen in intact cells. Mixing experiments with lysates isolated from untreated or treated cells provide no evidence for a translational inhibitor. However, experiments analyzing the incorporation of [35S]methionine and [35S]Met-tRNAf into initiation complexes showed that while the level of labeled 40S initiation complex in lysates from treated cells was the same or higher than in control lysates, the rate or efficiency of formation of the 80S initiation complex was inhibited. These results imply that the transcriptional inhibitors do not affect the level or charging of the initiation tRNAMet, the activity of the eIF-2 initiation factor needed for ternary complex formation, and the availability of active 40S ribosomal subunits. Thus, this site of action is different from that observed in other translational control systems such as the hemin response in reticulocytes and the interferon-induced translation inhibition in virally infected cells. This effect may reflect the cell's coordination of nuclear transcription and cytoplasmic translation.