The matrix (M) protein of vesicular stomatitis virus (VSV) binds the nucleocapsid to the cytoplasmic surface of the host plasma membrane during virus assembly by budding. It also condenses the nucleocapsid into a tightly coiled nucleocapsid-M protein complex that appears to give the virion its bullet-like shape. As described here, temperature-sensitive (ts) M mutants produced two classes of membrane-containing extracellular particles at the nonpermissive temperature. These could be distinguished by sedimentation in sucrose gradients and by electron microscopy. One class contained nucleocapsids and envelope glycoprotein, but very little M protein. The other class was devoid of nucleocapsids. Most of these particles were spherical or pleiomorphic in shape as determined by electron microscopy. Expression of wild-type (wt) M protein from plasmid DNA using the vaccinia/T7 virus system did not enhance the incorporation of nucleocapsids into extracellular particles from cells coinfected with the ts M mutants but did enhance the incorporation of M protein into these particles. Electron microscopy showed that wt M protein served to impart the bullet-like shape typical of VSV virions to what would otherwise be spherical or pleiomorphic virus-like particles. These data suggest that there are two distinct processes in VSV envelope biogenesis. One process involves envelopment of the nucleocapsid and can be accomplished by the ts M mutants at the nonpermissive temperature, albeit at a low level compared to wt VSV. The other process involves conversion of virion components into the bullet-like shape and requires a function provided by wt M protein.