Adenovirus (Ad) type 5 DNA packaging is initiated in a polar fashion from the left end of the genome. The packaging process is dependent upon the cis-acting packaging domain located between nucleotides 194 and 380. Seven A/T-rich repeats have been identified within this domain that direct packaging. A1, A2, A5, and A6 are the most important repeats functionally and share a bipartite sequence motif. Several lines of evidence suggest that there is a limiting trans-acting factor(s) that plays a role in packaging. Two cellular activities that bind to minimal packaging domains in vitro have been previously identified. These binding activities are P complex, an uncharacterized protein(s), and chicken ovalbumin upstream promoter transcription factor (COUP-TF). In this work, we report that a third cellular protein, octamer-1 protein (Oct-1), binds to minimal packaging domains. In vitro binding analyses and in vivo packaging assays were used to examine the relevance of these DNA binding activities to Ad DNA packaging. The results of these experiments reveal that COUP-TF and Oct-1 binding does not play a functional role in Ad packaging, whereas P-complex binding directly correlates with packaging function. We demonstrate that P complex contains the cellular protein CCAAT displacement protein (CDP) and that full-length CDP is found in purified virus particles. In addition to cellular factors, previous evidence indicates that viral factors play a role in the initiation of viral DNA packaging. We propose that CDP, in conjunction with one or more viral proteins, binds to the packaging sequences of Ad to initiate the encapsidation process.