The effects of inhibitors of protein synthesis upon transcription have been re-examined. Cycloheximide (1 microgram/ml) inhibits incorporation of uridine into RNA of P1798.S20 lymphosarcoma cells. Filter hybridization studies indicate that labeling of pre-rRNA is inhibited 60-80% after 1 h and quantitative S1 nuclease mapping reveals a corresponding decrease in the amount of cellular pre-rRNA. Cycloheximide also inhibits labeling of 5 S RNA and tRNA, but incorporation of uridine into poly(A+) RNA is unaffected. Transcription experiments carried out in nuclei from cycloheximide-treated cells indicate that the inhibitor causes a selective decrease in the activity of RNA polymerases I and III. Cell-free extracts from P1798.S20 were used to transcribe the cloned mouse rRNA gene, Syrian hamster 5 S RNA gene, and the Drosophila tRNAArg gene. Extracts from cycloheximide-treated cells were inhibited in this respect. Transcription of rRNA and 5 S RNA genes was inhibited by 90% after 2 h and 50% inhibition occurred within 20-30 min. Transcription of the tRNA gene was inhibited 75% after 2 h with a half-time of approximately 1 h. Inhibition was due neither to a direct effect of cycloheximide nor to the presence of nucleases or diffusible inhibitors of transcription. Moreover, transcription of rDNA in extracts from cycloheximide-treated cells could be restored by the addition of a partially purified initiation factor preparation. The data indicate that inhibition of protein synthesis results in rapid depletion of transcription factors that are required for initiation by RNA polymerases I and III. Among these is the glucocorticoid-regulated rDNA initiation factor designated TFIC.