The fate of adenovirus type 5r after infection of HeLa cells has been studied with the help of purified labeled virus preparations. The infection process starts with the adsorption of virions on the cell surface followed by penetration into the cells. Shortly after penetration, the particles inside the cells have a similar appearance as intact virions. However, a small amount of protein must have been lost, as concluded from radioactivity measurements and from the change in buoyant density (1.35 g/cm(3) instead of 1.34 g/cm(3), the value for intact virions). Evidence is presented that antigen B is absent from the 1.35-particles. After the transformation of virions into 1.35-particles, further uncoating leads to the formation of DNase-sensitive DNA-protein complexes. The proteins released during the uncoating show a buoyant density of 1.31 g/cm(3) and are almost completely insoluble in acid. No extensive breakdown of protein can be detected. The processes of attachment, penetration, and uncoating are not inhibited by prevention of de novo protein synthesis, as could be shown with cycloheximide.