Highly purified, (32)P-labeled type 5 adenovirus was employed to study "uncoating" of viral deoxyribonucleic acid (DNA)-defined as the development of sensitivity to deoxyribonuclease. Viral infectivity and radioactivity adsorbed to KB cells at the same rate, and significant amounts of (32)P did not elute from cells throughout the eclipse period. Kinetic studies of viral penetration, eclipse of infectivity, and uncoating of viral DNA indicated that the three events were closely related temporally, that the rates of each were similar, and that they were completed within 60 to 90 min after infection. Viral penetration, eclipse, and uncoating proceeded normally under conditions which blocked protein synthesis, but they did not occur at 0 to 4 C. Neither viral DNA nor viral protein was degraded to acid-soluble material during the eclipse period. The nature of adenovirus DNA was studied after it was converted intracellularly from deoxyribonuclease-resistant to deoxyribonuclease-susceptible. Intact virions centrifuged in sucrose gradients had a sedimentation coefficient of approximately 800, and viral DNA sedimented as a particle of about 30S. Infection of KB cells with purified (32)P-labeled virus yielded deoxyribonuclease-susceptible viral nucleic acid which was in particles with sedimentation coefficients of 350 to 450S, i.e., greater than 10 times faster than DNA obtained from purified virions which had been disrupted by exposure to pH 10.5. When the DNA from disrupted virions was mixed with cell lysates, its sedimentation characteristics were essentially unchanged by the presence of cellular material.