An aspect of the regulation of early adenovirus gene expression has been studied by measuring the rates of transcription of several viral transcription units during infection by wild-type (WT) adenovirus type 5 and a temperature-sensitive mutant, H5ts125. It has previously been shown that two transcription units (regions 2 and 4) are subject to negative control. During the course of early infection, transcription of regions 2 and 4 increased to a maximal rate and then declined. The decline from each of these transcription units appeared to be a specific shutoff of transcription, because the transcription of another early transcription unit (region 1A) remained constant during early infection and, in the absence of protein synthesis, the apparent shutoff of region 2 and region 4 transcription did not occur. At the nonpermissive temperature for the mutant, the kinetics of transcription of early region 2 were identical to the kinetics of transcription of WT. Thus, the repression of transcription of region 2 occurred in H5ts125-infected cells as well as in WT-infected cells. Region 4 transcription, however, was not repressed during H5ts125 infection at the nonpermissive temperature. After a maximal rate of transcription was reached, this rate was maintained throughout the course of the experiment. Furthermore, the repression of region 4 transcription appears to be the result of a block of initiation of transcription rather than the result of an inducement of premature termination of transcription. Therefore, it can be concluded that a product of the adenovirus region 2 gene, which is the site of the mutation for H5ts125, is responsible for the specific shutoff of region 4 transcription.