A previous analysis of naturally occurring defective interfering (DI) RNA genomes of the prototypic paramyxovirus simian virus 5 (SV5) indicated that 113 bases at the 3' terminus of the antigenome were sufficient to direct RNA encapsidation and replication. A nucleotide sequence alignment of the antigenomic 3'-terminal 113 bases of members of the Rubulavirus genus of the Paramyxoviridae family identified two regions of sequence identity: bases 1 to 19 at the 3' terminus (conserved region I [CRI]) and a more distal region consisting of antigenome bases 73 to 90 (CRII) that was contained within the 3' coding region of the L protein gene. To determine whether these regions of the antigenome were essential for SV5 RNA replication, a reverse genetics system was used to analyze the replication of copyback DI RNA analogs that contained a foreign gene (GL, encoding green fluorescence protein) flanked by 113 5'-terminal bases and various amounts of SV5 3'-terminal antigenomic sequences. Results from a deletion analysis showed that efficient encapsidation and replication of SV5-GL DI RNA analogs occurred when the 90 3'-terminal bases of the SV5 antigenomic RNA were retained, but replication was reduced approximately 5- to 14-fold in the case of truncated antigenomes that lacked the 3'-end CRII sequences. A chimeric copyback DI RNA containing the 3'-terminal 98 bases including the CRI and CRII sequences from the human parainfluenza virus type 2 (HPIV2) antigenome in place of the corresponding SV5 sequences was efficiently replicated by SV5 cDNA-derived components. However, replication was reduced approximately 20-fold for a truncated SV5-HPIV2 chimeric RNA that lacked the HPIV2 CRII sequences between antigenome bases 72 and 90. Progressive deletions of 6 to 18 bases in the region located between the SV5 antigenomic CRI and CRII segments (3'-end nucleotides 21 to 38) resulted in a approximately 25-fold decrease in SV5-GL RNA synthesis. Surprisingly, replication was restored to wild-type levels when these length alterations between CRI and CRII were corrected by replacing the deleted bases with nonviral sequences. Together, these data suggest that a functional SV5 antigenomic promoter requires proper spacing between an essential internal region and the 3' terminus. A model is presented for the structure of the 3' end of the SV5 antigenome which proposes that positioning of CRI and CRII along the same face of the helical nucleocapsid is an essential feature of a functional antigenomic promoter.