The adenovirus 294R protein of E4 ORF 6 forms a physical and functional complex with the 496R protein product of E1b. The E4 294R ORF 6 protein also functions in parallel with the E4 116R ORF 3 protein in viral late protein synthesis. We have examined the roles of these three proteins and the protein complex in viral late protein synthesis and late message metabolism, by comparing the phenotypes of E4 294R-, E4 116R-, and E1b 496R- mutants to those of a series of double mutants. Our data indicate that the 294R and 116R proteins act in parallel to permit the accumulation of normal levels of unprocessed late viral RNA in the nucleus of infected cells. Both 294R and 496R function in parallel with the 116R protein in viral nuclear RNA accumulation, but do so to different degrees, suggesting that 294R and 496R may have roles apart from the functional complex in mediating accumulation of viral messages in the nucleus, or that they have nonequivalent roles within the complex. Our results are also consistent with a role for the 496R/294R protein complex in mediating efficient transport of late messages from the nucleus to the cytoplasm and/or in maintaining the stability of those messages on reaching the cytoplasm, as suggested previously (S. Pilder, M. Moore, J. Logan, and T. Shenk, 1986, Mol. Cell. Biol. 6, 470-476). Finally the 116R protein seems to act in parallel with the complex to permit normal viral DNA replication.