The mechanism of the transcriptional turn-off was studied for those species of mRNA in T4 infection which specify early phage proteins. Total synthesis of these early RNA species was determined by short-time labeling with uracil-5-(3)H after the infection of Escherichia coli B with DNA-negative, conditional lethal mutants, which produce no late mRNA under nonpermissive conditions. With both amber and temperature-sensitive mutants a decrease in incorporation into early RNA by more than 90% was observed within 15 min after infection. Analysis by thin-layer chromatography showed uracil-5-(3)H to enter nucleotide pools throughout the infection cycle. The observed decrease in early RNA synthesis was dependent on protein synthesis, since it could be prevented by chloramphenicol. By varying the time of chloramphenicol addition, the effect of this regulatory protein(s) was found to appear at 3 to 4 min and reached its full extent at 10 to 15 min after infection. The turn-off of transcription of a defined early gene was studied by 5-fluorouracil (FU) rescue of an early, DNA-negative, amber mutant (gene 1, deoxynucleotide kinase) at different times after infection. DNA synthesis could be rescued by FU addition early after infection, but not after 12 min. 5-fluorouracil was efficiently taken up into the nucleotide pools also at late times, as shown by thin-layer chromatography. After incubation in the presence of chloramphenicol from 3 min after infection, DNA synthesis could be rescued as late as 45 min after infection. Thus, when protein synthesis was blocked during the early period of infection, the shut-off of transcription of the studied early gene was prevented.