To investigate further the contribution of the adenovirus type 5 (Ad5) E1B 55-kDa protein to genome replication, viral DNA accumulation was examined in primary human fibroblasts and epithelial cells infected with Ad5 or the E1B 55-kDa-null mutant Hr6. Unexpectedly, all cell types were observed to contain a significantly higher concentration of entering Hr6 than of Ad5 DNA, as did an infectious unit of Hr6. However, the great majority of the Hr6 genomes were degraded soon after entry. As this unusual phenotype cannot be ascribed to the Hr6 E1B frameshift mutation (J. S. Chahal and S. J. Flint, J. Virol. 86:3064-3072, 2012), the sequences of the Ad5 and Hr6 genomes were compared by using high-throughput sequencing. Seven previously unrecognized mutations were identified in the Hr6 genome, two of which result in substitutions in virion proteins, G315V in the preterminal protein (preTP) and A406V in fiber protein IV. Previous observations and the visualization by immunofluorescence of greater numbers of viral genomes entering the cytosol of Hr6-infected cells than of Ad5-infected cells indicated that the fiber mutation could not be responsible for the low-infectivity phenotype of Hr6. However, comparison of the forms of terminal protein present in purified virus particles indicated that the production of mature terminal protein from a processing intermediate is impaired in Hr6 particles. We therefore propose that complete processing of preTP within virus particles is necessary for the ability of viral genomes to become localized at appropriate sites and persist in infected cells.