The functional template for transcription of vesicular stomatitis virus (VSV) RNA is a ribonucleoprotein particle (nucleocapsid) consisting of the negative-strand sense genomic RNA completely encapsidated by the viral nucleocapsid (N) protein. As an approach to create nucleocapsids in vitro, we demonstrate here the specific encapsidation by purified N protein of in vitro-synthesized RNA sequences representing the 5' end of both the negative- and positive-strand VSV genome-length RNAs. As few as 19 nucleotides from the 5'-end of positive-strand RNA allowed maximal encapsidation, although the 5' terminal 10 nucleotides would allow partial (50%) encapsidation. Sequences downstream of the binding site can be of any origin. Specific encapsidation of VSV sequences was dependent on the presence of uninfected cell cytoplasmic extracts or poly(A). The synthetic nucleocapsids have the properties of RNase resistance and a buoyant density typical of wild-type VSV nucleocapsids. We have encapsidated a synthetic virionlike RNA species which contained just the terminal sequences of the virion RNA: the N encapsidation signal from the 5' end and the leader gene from the 3' end. This assembled nucleocapsid could function in vitro as a transcription template for the VSV RNA polymerase.