We have reconstituted synthetic VSV nucleocapsids from synthetic RNA and purified VSV N protein which are active as templates for transcription by the viral RNA polymerase in vitro. Utilizing progressively shorter RNAs in this system, we found that nucleocapsids containing just the 3' 22 nucleotides of either (-) or (+) strand viral RNA served as good transcription templates. Of this sequence, optimal transcription required the 15-17 3'-terminal nucleotides as a promoter. We showed with two VSV serotypes that the 3'-terminal nucleotides, UGC, were absolutely essential for transcription of the synthetic nucleocapsid by added RNA polymerase. Addition of extra nucleotides to the normal 3' end of nucleocapsid RNA totally abolished activity. The requirements for the nucleotides from positions 4 to 17 from the 3' end were less stringent, since individual changes in this region had variable effects on transcription, from partial inhibition to stimulation. The characterization of the transcription products synthesized from a variety of nucleocapids showed both full-length products and short (8-12 nucleotides) products which initiated directly at the 3' end but prematurely terminated. The VSV RNA polymerase was capable of adding one or more nontemplated bases to the 3' end of the product RNA depending on the nucleocapsid RNA sequence and the presence or absence of 5' triphosphates on the RNA.