Adenovirus prevents host cell protein synthesis during its late phase of replication in large part by causing the underphosphorylation of translation initiation factor eIF-4E, a component of initiation factor eIF-4F (cap-binding protein complex). Late adenovirus mRNAs are preferentially translated because they possess a reduced requirement for eIF-4F. This study continues the characterization of the mechanism by which adenovirus inhibits cellular protein synthesis. First it is shown that adenovirus blocks the addition of phosphate to eIF-4E rather than enhancing its removal, establishing that the virus impairs a signalling pathway or protein kinase activity involved in eIF-4E phosphorylation. It is then shown that shutoff of cell protein synthesis and translation of late viral mRNAs are uncoupled, in that shutoff actually occurs a short time (1 to 3 h) after late adenovirus mRNAs are already undergoing translation. Finally, by using a variety of genetic mutants stalled at different stages in the viral life cycle, it was found that dephosphorylation of eIF-4E and inhibition of cell translation are not caused by early adenovirus gene products acting at late times or by events related to viral DNA replication. Instead, it is shown that inhibition of eIF-4E phosphorylation and cell translation are mediated upon activation of the viral major late transcription unit. These and other results presented indicate that the adenovirus signal which induces eIF-4E dephosphorylation and shutoff of cell protein synthesis is linked either to an activity of one or more late viral polypeptides, to double-stranded RNA produced by opposition of the early and late viral transcription units, or to both.