The early and the late genes of simian virus 40 (SV40) are transcribed in opposite directions from a shared promoter region. The 72- and the 21-bp repeat regions of the SV40 genome contain the transcriptional enhancer and six copies of the Sp 1-binding GGGCGG box, respectively. SV40 mutants lacking various parts of these regions were examined in COS cells to determine the importance of these sequences for transcription in each direction. We made the following observations. (i) The 72-bp repeat region was required for efficient transcription of both the early and the late genes. (ii) The 21-bp repeat region was required for efficient early-gene transcription, but not for efficient late-gene transcription; however, it was able to supply some late-promoter activity when the 72-bp repeat region was missing. (iii) The ability of either of these regions to promote transcription was gradually reduced as the number of promoter elements within each was decreased. (iv) Mutations in these regions always decreased early-gene transcription more than late-gene transcription. These results indicate that both regions are made up of multiple bidirectional promoter elements, but that the 72-bp repeat region is more effective at inducing transcription than the 21-bp repeat region. Since each region can also (i) satisfy a need for promoter elements in the replication of viral DNA and (ii) induce a region of open chromatin, we conclude that the promoter elements within the enhancer and the GGGCGG boxes probably provide similar functions.