Measles virus nucleoprotein encoded from the vaccinia virus genome assembles into nucleocapsids similar in many respects to those observed during a natural measles virus infection. The influence of the measles virus phosphoprotein on nucleocapsid assembly has been studied using a vaccinia virus recombinant encoding both the nucleoprotein and the phosphoprotein. Infection of cells with the virus recombinant resulted in the formation of cytoplasmic inclusions in which the nucleoprotein and the phosphoprotein colocalized. Electron microscopic examination suggested that these inclusions contained characteristic nucleocapsid filaments. The buoyant density of nucleocapsids assembled in the presence of the phosphoprotein was found to be slightly higher than that of nucleocapsids assembled in its absence. Furthermore, the phosphoprotein partially inhibited the formation of nucleocapsids, a process which was extremely efficient when the nucleoprotein was expressed alone. Analysis of the nucleic acid content of nucleocapsids showed that they packaged heterologous RNA into a micrococcal nuclease-resistant form. These experiments demonstrate that the measles virus phosphoprotein regulates the efficiency with which the nucleoprotein assembles into nucleocapsids and the structural conformation they acquire.