A functional RNA replication promoter for the paramyxovirus simian virus 5 (SV5) requires two essential and discontinuous elements: 19 bases at the 3' terminus (conserved region I) and an 18-base internal region (conserved region II [CRII]) that is contained within the coding region of the L protein gene. A reverse-genetics system was used to determine the sequence requirements for the internal CRII element to function in RNA replication. A series of copyback defective interfering (DI) RNA analogs were constructed to contain point mutations in the 18 nucleotides composing CRII, and their relative replication levels were analyzed. The results indicated that SV5 DI RNA replication was reduced by substitutions for two CG dinucleotides, which in the nucleocapsid template are in the first two positions of the first two hexamers of CRII nucleotides. Substitutions for other bases within CRII did not reduce RNA synthesis. Thus, two consecutive 5'-CGNNNN-3' hexamers form an important sequence in the SV5 CRII promoter element. The position of the CG dinucleotide within the SV5 leader and antitrailer promoters was highly conserved among other members of the Rubulavirus genus, but this motif differed significantly in both sequence and position from that previously identified for Sendai virus. The possible roles of the CRII internal promoter element in paramyxovirus RNA replication are discussed.