Single-stranded fragments of adenovirus type 2 DNA were isolated from infected KB cells under conditions which retarded reassociation of complementary sequences but did not denature native viral DNA. Of the total intracellular, virus-specific DNA labeled during a 1-h pulse with tritiated thymidine begining 15 h after infection, about 20% was single stranded when fractionated on hydroxylapatite. This DNA shifted predominantly to the double-stranded fraction on hydroxylapatite during an extended chase incubation, suggesting that it may represent single-stranded DNA in replicating intermediates. Furthermore, the single-stranded DNA annealed nearly equally to both strands of the adenovirus genome. These findings indicate that at least portions of both complementary strands of adenovirus type 2 DNA are exposed as single strands during the period of viral DNA synthesis.