Infectious defective interfering (DI) particles of the negative-stranded RNA virus vesicular stomatitis virus (VSV) have been recovered from negative-sense transcripts of a plasmid that contains a full-length cDNA derived from the DI-T particle genome. In order to determine the cis-acting sequences necessary for RNA replication, encapsidation, and budding and to approximate the minimal size of RNA that can be packaged into infectious particles, we constructed a series of internal deletions in the DI cDNA to generate plasmids that could be transcribed to yield RNAs which ranged in size from 2209 nucleotides down to 102 nucleotides. All the deletion plasmids retained at least 36 nucleotides from the 5'-terminus and 51 nucleotides from the 3'-terminus of the DI genome. In cells expressing the five VSV proteins, the deleted DI RNAs were examined for their ability to be encapsidated, to replicate, and to bud to produce infectious DI particles. An RNA as small as 191 nucleotides, which contained 46 nucleotides from the 5'-end and 145 nucleotides from the 3'-end of the DI genome was encapsidated, replicated, and budded at least as efficiently as the full-length wild-type DI RNA. In contrast, a 102-nucleotide RNA that contained only the 51 nucleotides from the 5'-end of the DI RNA and its perfect 51-nucleotide complement at the 3'-end replicated poorly and failed to bud infectious DI particles. However, an RNA with an insertion of 1499-nucleotide "stuffer" sequences of non-VSV origin between the two 51-nucleotide complementary termini not only replicated but also budded infectious particles. These data show that the signals necessary for RNA encapsidation, replication, and packaging into infectious DI particles are contained within the 5'-terminal 36 nucleotides and the 3'-terminal 51 nucleotides of the DI RNA genome. Furthermore, the results show that a heterologous sequence can be replicated and packaged into infectious particles if it is flanked by the DI RNA termini.