All mRNAs expressed from the adenovirus major late transcription unit have a common, 201-nucleotide-long 5' leader sequence, which consists of three short exons (the tripartite leader). This leader has two variants, either with or without the i-leader exon, which, when present, is spliced between the second and the third exons of the tripartite leader. Previous studies have shown that adenovirus early region 4 (E4) encodes two proteins, E4 open reading frame 3 (E4-ORF3) and E4-ORF6, which are required for efficient expression of mRNAs from the major late transcription unit. These two E4 proteins appear to have redundant activities, and expression of one has been shown to be sufficient for efficient major late mRNA accumulation during a lytic virus infection. In this report, we provide evidence that E4-ORF3 and E4-ORF6 both regulate major late mRNA accumulation by stimulating constitutive splicing. Moreover, we show that the two proteins have different effects on accumulation of alternatively spliced tripartite leader exons. In a DNA transfection assay, E4-ORF3 was shown to facilitate i-leader exon inclusion, while E4-ORF6 preferentially favored i-leader exon skipping. In addition, E4-ORF3 and E4-ORF6 had the same effects on accumulation of alternatively spliced chimeric beta-globin transcripts. This finding suggests that the activities of the two proteins may be of more general relevance and not restricted to splicing of major late tripartite leader-containing pre-mRNAs. Interestingly, E4-ORF6 expression was also shown to stimulate i-leader exon skipping during a lytic virus infection.