HIV-1 Rev transactivator is readily phosphorylated at separate regions by protein kinase CK2 and MAP kinase. Protein kinase CK1 cannot replace CK2 as phosphorylating agent and cdc2 only slowly phosphorylates Rev at one of the two sites affected by MAP kinase. Mutational analysis shows that Ser-8 and, to a lesser extent, Ser-5 are phosphorylated by CK2. In contrast, a mutation (R14TV-->EED) which suppresses Rev activity dramatically enhances Rev phosphorylation either in vitro by CK2 or in vivo, suggesting that phosphorylation by CK2 could play a role in Rev down-regulation.