The association of M protein and the nucleocapsid (N) protein of vesicular stomatitis virus (VSV) with the cytoplasmic surface of plasma membranes prepared from infected cells was examined by double label immunofluorescence. M protein in association with the cytoplasmic surface of the plasma membrane was distributed in two distinct labeling patterns. Punctate labeling of M protein in the plasma membrane was observed in association with corresponding labeling for the nucleocapsid protein. Diffusely labeled M protein was distributed in areas of the plasma membrane that were devoid of any detectable labeling for the nucleocapsid protein. Similar results were obtained with two different cell types at 4 h and later times postinfection. The diffuse label for M protein was present in membranes prepared from cells infected with a temperature-sensitive M protein mutant at the nonpermissive temperature, but neither the punctate label for M protein nor labeling for the nucleocapsid protein was observed. Upon shift to permissive temperature, both the punctate label for M protein and labeling for the nucleocapsid protein began to reappear in membranes prepared from cells infected with the M protein mutant. These results indicate that M protein can associate with the plasma membrane without prior binding to nucleocapsids and that association of functional M protein with the plasma membrane is required for the stable association of nucleocapsids with the membrane during the process of viral budding.