The matrix (M) protein of vesicular stomatitis virus (VSV) plays a central role in virus assembly by binding the nucleocapsid core to the viral envelope during the budding process. A small percentage of M protein molecules are phosphorylated in vivo, but the role of phosphorylation in M protein function is unknown. Using limited proteolysis, we previously determined the sites of in vivo phosphorylation for VSV M protein to be Thr 31 (and possibly Ser 32) and a site N-terminal to position 19 (Ser 2, Ser 3, or Ser 17) (P. E. Kaptur et al., J. Virol. 66, 5384-5392, 1992). M protein mutants were constructed using site-directed mutagenesis by substituting Ala for Ser or Thr at these sites in the M gene of the San Juan strain of VSV. One mutant had substitutions at the major in vivo phosphorylation site(s) at positions 31 and 32 (M31.32) while two others had additional substitutions at positions 2 and 3 (M2.3.31.32) or at position 17 (M17.31.32). Mutant M proteins were expressed in BHK cells using the vaccinia/T7 system, radiolabeled with 32Pi, and then analyzed for 32P content by PAGE and autoradiography. The data show that the site of phosphorylation near the N-terminus is at Ser 2 or 3 and not Ser 17. Further, Ser 38 was not phosphorylated. Mutation of the major phosphorylation site enhanced phosphorylation at alternative sites in the M protein C-terminal to amino acid 43 and at Ser residues 2 and 3. Mutant M proteins were tested for their ability to complement growth of the temperature-sensitive M protein mutant virus tsO23 at the nonpermissive temperature. Mutant M2.3.31.32 was further tested for its ability to assemble into VSV-defective interfering (DI) particles, using a replication system in which the DI genome and all five VSV proteins were expressed from plasmid DNA. Assembly of tsO23 virions or DI particles in the presence of mutant M proteins was similar to that observed for wild-type M proteins. These data indicate that phosphorylation of M protein at the major in vivo sites is not essential for virus assembly.