The cis-acting signals found at the 3' ends of the genomic and antigenomic RNAs are a major factor determining the level of paramyxovirus RNA replication from each promoter. Using a minigenome system that reconstitutes SV5 RNA synthesis from cDNA-derived components, we show here that the genomic promoter (GP) for the paramyxovirus SV5 directs RNA replication approximately 14-fold lower than that seen from the antigenomic promoter (AGP). The goal of this study was to identify cis-acting signals responsible for differential levels of RNA replication from the SV5 GP and AGP. We have previously shown that the SV5 AGP contains three sequence-dependent elements (CRI, CRII, and Region III) that are separated by sequence-independent spacer regions. Minigenomes containing chimeric promoters were constructed to test the hypothesis that transfer of discrete cis-acting AGP elements to the GP could confer higher replication properties to the GP. Minigenomes containing a substitution of the AGP CRI, CRII, or Region III elements alone in place of the corresponding GP sequences did not show enhanced levels of RNA replication. However, transfer of both the AGP 3' terminal CRI and Region III elements into the corresponding sites of the GP led to a minigenome which replicated to approximately 40% of the levels seen with the AGP. This enhanced RNA replication from the GP was further increased up to AGP levels by also including the intervening AGP segment (bases 20-50) located between CRI and Region III. Importantly, transfer of nonviral sequences in place of GP bases 20-50 also increased RNA replication to levels approaching that of the AGP, but only in the context of the AGP CRI and Region III substitutions. These data indicate that differential levels of RNA replication from the SV5 GP and AGP are due to a combination of positive-acting signals in the AGP (CRI and Region III) and a negative-acting signal in the GP (bases 20-50). Possible functions for the SV5 promoter elements in determining RNA replication levels are proposed.