We have investigated the contribution of each of the two adenovirus type 5 (Ad5) major early region 1b (E1b) proteins in cell transformation and in lytic infection. An Ad5 E1 plasmid, in which the reading frame for the 19-kDa E1b protein was abolished by a stop codon close to the initiation codon, transformed primary baby rat kidney (BRK) cells with an efficiency of about half of that of a wild type Ad5 E1 plasmid, whereas a plasmid with a mutation in the gene for the 58-kDa E1b protein transformed the same primary cells with only one-third of the wild type efficiency. Plasmids containing region E1a only or a plasmid carrying mutations in the genes for major E1b proteins all transformed primary cells with an efficiency of approximately 5% of wild type. To test the effect of the E1b mutations in virion-mediated cell transformation, the mutant E1b regions were introduced into intact viral genomes by overlap recombination and were subsequently used in a transformation assay on BRK cells. The 19 and 58-kDa mutant viruses were found to transform BRK cells with 11 and 25% of the efficiency of wild type virus, respectively. These results suggest that the 19-kDa E1b protein is essential for virus-mediated cell transformation, in agreement with results of others, but not for plasmid-mediated cell transformation. In lytic infection, the 19-kDa mutant virus was some 30-fold reduced in yield on HeLa cells, whereas the 58-kDa mutant virus was 3000-fold reduced in its ability to grow on HeLa cells at low multiplicity of infection, but showed a marked multiplicity-dependent leakiness. The 58-kDa mutant virus was not defective when its growth was assayed on human embryonic kidney (HEK) cells. This may indicate that cellular proteins are expressed in HEK cells that are functionally homologous to the 58-kDa E1b protein.