In order to define the functions of human adenovirus type 12 (Ad12) early region 1 (E1) products in lytic infection and oncogenic transformation we have isolated and phenotypically characterized a set of host-range (hr) mutants of this serotype. These mutants grow efficiently upon HER3 cells, which contain and express type 12 E1 genes, but are restricted for growth upon A549 carcinoma and HeLa cells. Inter- and intratypic complementation analysis, marker-rescue mapping, and DNA sequence analysis have assigned some of the mutations to E1A sequence, and some to the reading frame encoding the E1B 54-kDa (482R) protein. Phenotypic analysis of the E1B mutants in particular has revealed some interesting, and in some cases surprising, findings relating to the roles of that protein in virus-cell interactions. This Ad12 gene product is required, either directly or indirectly, for efficient viral DNA replication in A549 and HeLa cells, unlike its counterpart in type 5 virus. Surprisingly, however, despite the severe defect in viral DNA replication, the synthesis of a few species of viral late proteins continues in cells infected by some of the E1B mutants. In contrast, none of these mutants brings about the inhibition of host-cell protein synthesis characteristic of wild-type virus infection, and with some E1B mutants no viral late proteins are made. Further, in a separate study reported elsewhere, we have demonstrated that the E1B 54-kDa product may also be involved, either directly or indirectly, in positive regulation of both E1A and E1B 19-kDa (163R) gene expression. The molecular and/or physiological bases for these various effects remain to be determined, but our initial results suggest that the E1B 54-kDa protein may carry out multiple regulatory functions during the viral life cycle.