IVa2 is an essential, multifunctional protein of adenovirus (Ad) supporting packaging of the viral genome into the capsid, assisting in assembly of the capsid, and activating Ad late transcription. A comparison of IVa2 protein sequences from different species of Adenoviridae shows conserved motifs associated with binding and hydrolysis of ATP (Walker A and B motifs). ATPases are essential proteins of bacteriophage packaging motors, and such activity may be required for Ad packaging. Results presented here show that the Ad2 IVa2 protein binds ATP in vitro and that sequences in the Walker A and B motifs are necessary for this activity.