Mutants with deletions in the control region of simian virus 40 (SV40) were tested for their ability to direct late transcription in a nuclear extract derived from HeLa cells. Primer extension analysis revealed that late SV40 transcription initiates predominantly at two sites in vitro, one of which corresponds to the major in vivo start site at nucleotide 325, while the other site is located at nucleotide 170. A series of 5',3', and internal deletions of the putative promoter region were used to define two distinct control elements that appear to function independently of each other and that are located upstream from each of the in vitro initiation sites. In addition, transcription from the initiation site at 325 is also influenced by GC-rich sequences (CCGCCC) found within a regulatory region that consists of two 21 bp perfect repeats and a third degenerate repeat located 250 bp upstream from the major late initiation site. These six upstream GC blocks, which lie directly upstream from the initiation site at 170, also affect transcription from this start site. Although these 21 bp repeats are known to be an important part of the SV40 early promoter, our findings suggest that they are also involved in modulating the levels of late transcription in vitro.