The genome of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) consists of two negative-sense, single-strand RNA segments designated L and S. Arenavirus genomes exhibit high sequence conservation at their 3' ends. All arenavirus genomes examined to date have a conserved terminal sequence element (3'-terminal 20 nucleotides [nt]) thought to be a highly conserved viral promoter. Terminal complementarity between the 5' and 3' ends of the L and S RNAs predicts the formation of a thermodynamically stable panhandle structure that could contribute to the control of RNA synthesis. We investigated these issues by using a transcription- and replication-competent minireplicon system. A series of overlapping deletions spanning the 3'-terminal 20-nt region of an LCMV minigenome (MG) was generated, and the mutant MGs were analyzed for their activity as templates for RNA synthesis by the LCMV polymerase. The minimal LCMV genomic promoter was found to be contained within the 3'-terminal 19 nt. Substitution of C for G at the last 3'-end nucleotide position in the MG resulted in nondetection of RNA transcription or replication, whereas the addition of a C at the 3' end did not have any significant affect on RNA synthesis mediated by the LCMV polymerase. All other mutations introduced within the 3'-terminal 19 nt of the MG resulted in undetectable levels of promoter activity. Deletions and nucleotide substitutions within the MG 5' end that disrupted terminal complementarity abolished chloramphenicol acetyltransferase expression and RNA synthesis mediated by the LCMV polymerase. These findings indicate that both sequence specificity within the 3'-terminal 19 nt and the integrity of the predicted panhandle structure appear to be required for efficient RNA synthesis mediated by the LCMV polymerase.