The adenovirus type 2 (Ad2) host range mutant Ad2hr400 grows efficiently in cultured monkey cells at 37°C, but is cold sensitive for plaque formation and late gene expression at 32.5°C. After nitrous acid mutagenesis of an Ad2hr400 stock, cold-resistant variants were selected in CV1 monkey cells at 32.5°C. One such variant, Ad2ts400, was also temperature sensitive (ts) for growth in both CV1 and HeLa cells. Marker rescue analysis has been used to show that the two phenotypes, cold resistant and ts, are due to two independent mutations, each of which resides in a different segment of the gene encoding the 72-kilodalton DNA binding protein (DBP). The cold-resistant mutation (map coordinates 63.6 to 66) is a host range alteration that enhances the ability of the virus to express late genes and grow productively in monkey cells at 32.5°C. The ts mutation is in the same complementation group and maps to the same segment of the DBP gene (map coordinates 61.3 to 63.6) as the well-characterized DBP mutant Ad5ts125. Like Ad5ts125, Ad2ts400 is unable to replicate viral DNA or to properly shut off early mRNA expression at the nonpermissive temperature. Two sets of experiments with Ad2ts400 suggest that DBP contains separate functional domains. First, when CV1 cells are coinfected at the nonpermissive temperature with Ad2 plus Ad2ts400 (Ad2 allows DNA replication and entry into, but not completion of, the late phase of infection), normal late gene expression and productive growth occur. Second, temperature shift experiments show that, although DNA replication is severely restricted at the nonpermissive temperature in ts400-infected monkey cells, late gene expression occurs normally. These results indicate that the DBP activity required for normal late gene expression in monkey cells is functional even when the DBP's DNA replication activity is disrupted.