A system for studying the in vitro replication of the RNA genomes of both wild-type vesicular stomatitis virus (VSV) and its defective interfering particle MS-T has been developed. After lysolecithin treatment of cells infected with VSV or VSV plus MS-T, a cell-free cytoplasmic extract is prepared which will support VSV mRNA synthesis and the synthesis of the 42S wild-type or 19S MS-T genome RNAs. The genome-length RNAs synthesized in vitro are assembled into RNase-resistant nucleocapsids. The level of 42S RNA synthesis in vitro (6-13% of total RNA synthesis) reflects the level of replication in vivo. Although the extracts of VSV-infected cells can also support the synthesis of VSV proteins, RNA replication is not dependent on de novo protein synthesis but utilizes the preformed soluble proteins present in the infected cell at the time the extract is prepared. The initiation of genomic RNA during in vitro replication can be demonstrated because detergent-disrupted, purified MS-T particles will replicate their RNA when added to either a total cytoplasmic extract from VSV-infected cells or the soluble protein fraction derived from such an extract.