The adenovirus type 5 mutants H5hr1 and H5dl101 contain modifications in the E1a gene affecting the 13S mRNA-encoded 289-amino acid polypeptide and exhibit a cold-sensitive transformation phenotype upon infection of cloned rat embryo fibroblast (CREF) cells. Transformed cell lines expressing solely E1a or E1a and E1b gene products derived from these viruses display enhanced anchorage-independent growth at 37 degrees C versus 32 degrees C and display a cytoskeletal architecture resembling untransformed fibroblastic CREF cells. In contrast, CREF cells transformed by H5wt or the E1a and E1b region of H5wt grow with similar efficiency in agar at 37 degrees C or 32 degrees C and exhibit an epithelioid morphology that is associated with an altered cytoskeleton. Regardless of the expression or presence of other viral early regions, including E1b, E2a, and E4 genes, specific CREF cell lines expressing an altered 289-amino acid protein and a wild-type 12S mRNA-encoded 243-amino acid protein were capable of inducing tumors in nude mice and in immunocompetent syngeneic Fischer rats. In sharp contrast, cells expressing a wild-type 289-amino acid protein were unable to induce tumors in either nude mice or syngeneic rats. The ability to induce tumors did not correlate with alterations in the pattern of viral DNA integration or differential expression of the E1a and E1b genes, nor was the tumor induction a consequence of unique properties of the immortal parental CREF cell line.