Casein kinase-II (CK-II) is a widely distributed protein kinase, which plays numerous roles in the regulation of transcription through modification of transacting transcription factors. Phosphorylation of vesicular stomatitis virus (VSV) P protein by CK-II was found to be both necessary and sufficient for transcriptional activation. Upon treatment of P by CK-II, activity was acquired faster (t1/2 = 3.7 min) than were total phosphates (t1/2 = 7.4 min). Stoichiometry was 2 mol phosphate/mol P, indicating activation by phosphorylation at either one or both of two independent sites. The sites were identified by substituting aspartate (D) residues at either S60 or T62, producing proteins that were partly active without phosphorylation, but were fully active at higher concentrations; CK-II added only a single phosphate group to each of these, and conferred full activity. P protein doubly substituted with D at S60 and T62 was fully active without phosphorylation, and was not a substrate for CK-II. Active P protein, whether CK-II treated or doubly substituted, was shown by gel filtration and crosslinking to exist as a discretely multimeric, probably tetrameric, structure. The singly substituted mutants were partly multimeric, becoming fully so after CK-II treatment. Phosphorylation by CK-II thus mediates the self-association of P into the multimeric, transcriptionally active form.