Many viral genes contain core promoters with two basal control elements, the TATA box and the pyrimidine-rich initiator (Inr). However, the molecular mechanisms involved in transcription initiation from composite core promoters (TATA(+) Inr(+)) containing Inr elements are unclear. The Rous sarcoma virus (RSV) long terminal repeat (LTR) contains a transcriptionally potent enhancer and core promoter composed of a TATA box and an Inr-like sequence, termed the transcription start site core (TSSC). Previously we demonstrated that the TSSC binds the multifunctional Inr-binding protein YY1. Here we present evidence that the TSSC also binds the multifunctional transcription factor TFII-I and that both TFII-I and YY1 are required for RSV LTR transcriptional activity. Gel shift assays using anti-TFII-I antibody show that TFII-I is present in a protein complex that specifically binds to the TSSC. Mutations in the TSSC that reduce TFII-I binding also reduce RSV LTR enhancer and promoter activity. Transient-transfection assays demonstrate that TFII-I transactivates the RSV LTR from ca. fourfold (basal) to ca. sevenfold (enhanced) in both human and natural host cell lines. Importantly, the activity of the TSSC element can be attributed to the binding activity of TFII-I and the YY1 protein, since mutation of each of these binding sites within the TSSC element abolishes all viral expression as demonstrated by transient-transfection assays. Taken together, these data demonstrate that expression of RSV viral mRNA is dependent on both TFII-I and YY1.