Most adenovirus-specific mRNAs expressed late after infection originate from a single transcription unit, the so-called major late transcription unit. All mRNAs expressed from this unit have in common a 201 nucleotide-long spliced tripartite leader segment attached to their 5' ends. Human adenovirus mutants that carry large deletions in early region 4 (E4) are severely defective in expression of nuclear and cytoplasmic RNA derived from the major late transcription unit. We have previously shown that E4 post-transcriptionally stimulates accumulation of tripartite leader containing mRNAs by a mechanism that requires an intron in the nuclear precursor RNA. To identify the E4 products responsible for this stimulatory effect on tripartite leader mRNA accumulation, we constructed CMV expression vectors encoding single E4 open reading frames (ORFs). By comparing the activity of these plasmids in a transient DNA cotransfection assay we could show that both the E4-ORF3 and E4-ORF6 proteins individually are able to stimulate mRNA accumulation from tripartite leader intron containing transcription units. Furthermore, this stimulatory activity was not dependent on coexpression of other viral gene products. These results are interesting since the same two E4 proteins have been shown to have interchangeable activities in lytic infection, and expression of one has been suggested to be sufficient to substitute for the whole E4 region for virus growth. Finally, we show that the absence of E4 expression during a virus infection results in abnormalities in tripartite leader assembly. This result suggests that E4 proteins may be required for efficient tripartite splicing also during a lytic virus infection.