Herpesvirus lytic DNA replication requires both the cis-acting element, the origin, and trans-acting factors such as virally encoded origin-binding protein and DNA replication enzymes. Recently, the origins of lytic DNA replication (ori-Lyt) in Kaposi's sarcoma-associated herpesvirus (KSHV) have been identified and a virally encoded bZip protein, K8, has been shown to specifically bind to the origin. To map cis-acting elements within KSHV ori-Lyt that are required for DNA replication function and to define the nature of K8 bZip protein binding to the origin, we constructed consecutive internal deletion mutations across the core domain of a KSHV ori-Lyt and tested them for DNA replication function in a transient replication assay. This mutagenesis study allowed the identification of four components within the ori-Lyt, and all were indispensable for ori-Lyt function. The first component contains eight CCAAT/enhancer binding protein (C/EBP) binding motifs that organize as four spaced C/EBP palindromes. Each palindrome contains two head-to-head CCAAT consensus motifs that are separated by a 13- or 12-bp space sequence. Substitution mutagenesis of these C/EBP motifs showed that these C/EBP palindromes are required for both K8 binding and ori-Lyt-dependent DNA replication. The second component is an 18-bp AT palindrome, which is essential for ori-Lyt function. The third component was determined to be a 32-bp previously unidentified sequence and is required for DNA replication. The last component consists of an open reading frame 50 (ORF50)/Rta responsive element (RRE) and a TATA box. We showed that the binding of an ORF50/Rta protein to the RRE was essential for ori-Lyt-dependent DNA replication. The presence of a functional RRE and a downstream TATA box suggested that this region serves as an ORF50/Rta-dependent promoter and a transcription event may be necessary for ori-Lyt-dependent DNA replication. Using a luciferase reporter system, we demonstrated that the region of the RRE and TATA box constitutes an ORF50/Rta-dependent promoter. Furthermore, a polyadenylated RNA of 1.4 kb was identified downstream of the promoter.