Polar packaging of adenovirus DNA into virions is dependent on the presence of cis-acting sequences at the left end of the viral genome. Our previous analyses demonstrated that the adenovirus type 5 (Ad5) packaging domain (nucleotides 194 to 358) is composed of at least five elements that are functionally redundant. A repeated sequence, termed the A repeat, was associated with packaging function. Here we report a more detailed analysis of the requirements for the selective packaging of Ad5 DNA. By introducing site-directed point mutations into specific A repeat sequences, we demonstrate that the A repeats represent cis-acting functional components of the packaging signal. Additional elements, located outside the originally defined packaging domain boundaries and that resemble the A repeat consensus sequence, also are capable of promoting the packaging of viral DNA. The cis-acting components of the packaging signal appear to be subject to certain spatial constraints for function, possibly reflecting a necessity for the coordinate binding of packaging proteins to these sites. In agreement with this idea, we present evidence that the interaction of a limiting trans-acting factor(s) with the packaging domain in vivo is required for efficient encapsidation of the Ad5 genome.