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Properties of type 7 adenovirions in lysosomes of HeLa cells were studied 12 hr postinfection. Viral particles were transferred to the lysosomes very quickly after initiation of penetration, i.e., after 10 min of incubation at 37 degrees. No morphological modification of the virion was detected for 6 hr postinfection. However, by 12 hr postinfection, the virion was no longer recognizable. Most of the virus remained infectious for 2 hr, whereas after 12 hr the infectivity was abolished. Soon after the adsorption of the virus on the cell membrane at 4 degrees, the viral DNA in the virion became sensitive to pancreatic DNase, and this sensitivity increased during the first 2 hr of incubation at 37 degrees. This result suggests that some modification in the architecture of the virion occurred before transfer to the lysosomes. The adenovirus 7 (Ad 7) DNA extracted from the lysosomes appeared intact for 6 hr postinfection and was found to cosediment at 34 S with the Ad 2 DNA marker. Comparable activities of free acid phosphatase were found in lysosomes isolated from uninfected control cells and from infected cells. In in vitro experiments, lysosomal acid DNase and pancreatic DNase were shown to degrade Ad 7 DNA at similar rates; however, in vivo, intralysosomal Ad 7 DNA was only partially sensitive to lysosomal DNase.