The Rous sarcoma virus (RSV) long terminal repeat (LTR) contains a transcriptionally potent enhancer and promoter that functions in a variety of cell types. Previous studies have identified the viral sequences required for enhancer activity, and characterization of these elements has provided insight into the mechanism of RSV transcriptional activity. The objective of this study was to better define the RSV LTR promoter by examining the transcription start site core (TSSC) region. Deletion of the TSSC resulted in complete loss of transcriptional activity despite the presence of a functional TATA box, suggesting that the TSSC is required for viral expression. Homologies within the TSSC to the DNA binding motif of YY1 suggested that it might regulate promoter activity. YY1 has been shown to regulate transcription in some cellular genes and viral promoters by binding to sites overlapping the transcription start site. Gel shift assays using YY1 antibody identified YY1 as one of three complexes that bound to the TSSC. Mutation of the YY1 binding site reduced RSV transcriptional activity by more than 50%, suggesting that YY1, in addition to other TSSC-binding factors, regulates RSV transcription. Furthermore, in vitro transcription assays performed with Drosophila embryo extract (devoid of YY1 activity) showed decreased levels of RSV transcription, while transient transfection experiments overexpressing YY1 demonstrated that YY1 could transactivate the RSV LTR approximately 6- to 7-fold. We propose that the TSSC plays a vital role in RSV transcription and that this function is partially carried out by the transcription factor YY1.