Summary In vitro assembly of human adenovirus type 5 was studied using virus precursor materials from extracts of HeLa cells infected in Eagle's complete and arginine-free medium. Radioisotope studies suggested that factors having assembly activities in vitro were present only in extracts of cells infected in complete medium. Extracts containing these assembly factors were added to extracts of cells infected in arginine-free medium and the mixtures, after incubation in the presence of stabilizing buffer solutions, contained particles of the same density as complete virus particles formed in vivo. The newly assembled products were DNase-resistant and had 1.5 to 2 log. greater infectivity in plaque assays than either of the two extracts alone or in unassembled mixtures. The factor(s) responsible for the assembly activity were found to be associated predominantly with the nuclear fraction of the infected cells and they were produced in greatest amounts in extracts taken at 12 to 16 hr after infection. Assembly activity of the extracts was lost when they were subjected to temperatures above 32°, trypsin treatment and fluorocarbon extraction before adding them to experimental assembly mixtures.