Specific mutation of the DNA binding protein (DBP) of human adenovirus types 2 and 5 can extend the host range of these viruses to simian cells. These mutations replace histidine at position 130 in the highly phosphorylated N-terminal domain of DBP with a potentially phophorylatable tyrosine residue. To investigate the possibility that alternative phosphorylation might contribute to the functional differences between wild type (wt) and host range (hr) DBP molecules, radiolabeled proteins were compared by partial proteolysis and tyrosine phosphorylation was analyzed. These studies confirmed the previous tentative assignment of a chymotrypsin-sensitive site at position 121 of DBP. No host range-specific tyrosine phosphorylation was detected, and no gross difference in the extent of phosphorylation between wt and hr DBP was observed. However, the cleaved N-terminal domains of wt and hr DBP exhibited different sensitivities to further chymotryptic digestion in vitro and different fragmentation patterns, suggesting that they might have different conformations. Such a difference could underlie the differing ability of these proteins to support Ad replication in simian cells.