SR proteins are a family of essential splicing factors required for early recognition of splice sites during spliceosome assembly. They also function as alternative RNA splicing factors when overexpressed in vivo or added in excess to extracts in vitro. SR proteins are highly phosphorylated in vivo, a modification that is required for their function in spliceosome assembly and splicing catalysis. Here we show that SR proteins purified from late adenovirus-infected cells are inactivated as splicing enhancer or splicing repressor proteins by virus-induced dephosphorylation. We further show that the virus-encoded protein E4-ORF4 activates dephosphorylation by protein phosphatase 2A of HeLa SR proteins and converts their splicing properties into that of SR proteins purified from late adenovirus-infected cells. Taken together, our results suggest that E4-ORF4 is an important factor controlling the temporal shift in adenovirus alternative RNA splicing. We conclude that alternative pre-mRNA splicing, like many other biological processes, is regulated by reversible protein phosphorylation.