The phosphorylation of the P protein of vesicular stomatitis virus by cellular casein kinase II (CKII) is essential for its activity in viral transcription. Recent in vitro studies have demonstrated that CKII converts the inactive unphosphorylated form of P (P0) to an active phosphorylated form P1, after phosphorylation at two serine residues, Ser-59 and Ser-61. To gain insight into the role of CKII-mediated phosphorylation in the structure and function of the P protein, we have carried out circular dichroism (CD) and biochemical analyses of both P0 and P1. The results of CD analyses reveal that phosphorylation of P0 to P1 significantly increases the predicted alpha-helical structure of the P1 protein from 27 to 48%. The phosphorylation defective double serine mutant (P59/61), which is transcriptionally inactive, possesses a secondary structure similar to that of P0. P1, at a protein concentration of 50 micrograms/ml, elutes from a gel filtration column apparently as a dimer, whereas both P0 and the double serine mutant elute as a monomer at the same concentration. Interestingly, unlike wild-type P1 protein, the P mutants in which either Ser-59 or Ser-61 is altered to alanine required a high concentration of CKII for optimal phosphorylation. We demonstrate here that phosphorylation of either Ser-59 or Ser-61 is necessary and sufficient to transactivate L polymerase although alteration of one serine residue significantly decreases its affinity for CKII. We have also shown that P1 binds to the N-RNA template more efficiently than P0 and the formation of P1 is a prerequisite for the subsequent phosphorylation by L protein-associated kinase. In addition, mutant P59/61 acts as a transdominant negative mutant when used in a transcription reconstitution assay in the presence of wild-type P protein.