The adenovirus origin of DNA replication is located within the terminal 51 bp of the viral genome and contains three recognizable domains: the minimal origin or "core" and binding sites for the cellular transcription factors NFI (CTF) and NFIII (oct-1, OTF-I). In vivo assays with a series of plasmids containing insertions between the "core" and NFI binding site revealed that a strict spatial arrangement of the NFI binding site relative to the "core" was required for efficient DNA replication. To determine if this strict positional constraint was a result of interactions between genome-bound proteins, we used the DNA-binding domain of NFI immobilized on Sepharose as an affinity matrix to examine binding of the adenovirus DNA polymerase and preterminal protein. Extracts from insect cells infected with baculoviruses expressing the polymerase or preterminal protein were passed over the NFI affinity matrix and bound proteins were eluted. Whereas preterminal protein passed through the column, the DNA polymerase was specifically retained. When extracts containing both preterminal protein and polymerase were passed over the NFI column, both proteins were retained because of the formation of DNA polymerase-preterminal protein heterodimers. Thus, interactions between the DNA binding domain of NFI and the DNA polymerase may serve to direct the DNA polymerase-preterminal protein heterodimer into a preinitiation complex that assembles at the adenovirus origin of DNA replication.