An adenovirus mutant, Ad2ts111, has previously been shown to be temperature sensitive for viral DNA replication in vivo and also to induce degradation of cellular DNA. Soluble nuclear extracts prepared from Ad2ts111-infected HeLa cells grown at either the permissive (32 degrees C) or the nonpermissive (39.5 degrees C) temperature are thermolabile for elongation but not for initiation of DNA replication in vitro. Adenovirus single-stranded-DNA-binding protein purified from wild-type-infected cells can complement these extracts at the restrictive temperature in vitro. The DNA-binding protein synthesized in Ad2ts111-infected cells is stable at the nonpermissive temperature and is phosphorylated, as is the wild-type protein. In contrast, the mutant DNA-binding protein synthesized in Ad5ts125-infected cells is unstable. Ad2ts111 and Ad5ts125 do not complement each other for virus growth in vivo. These results suggest that Ad2ts111 contains a mutation in the DNA-binding protein that affects viral DNA synthesis. Finally, we demonstrated that, unlike viral DNA synthesis, the induction of cellular DNA degradation in Ad2ts111-infected cells is not temperature sensitive and that this phenotype is a result of a mutation in early region 1 on the virus genome. Thus, the two phenotypes displayed in Ad2ts111-infected cells, namely, the temperature-sensitive replication of viral DNA and the degradation of cell DNA, are the result of two separate mutations.