A reconstituted transcription system composed of the Rift Valley fever phlebovirus (Bunyaviridae family) proteins L and N expressed via recombinant vaccinia viruses and an S-like model RNA containing the CAT gene in the antisense orientation, has been described previously by Lopez et al. (J. Virol., 1995, 69, 3972-3979). We extended the use of this in vivo system to determine the sequence at the 3' end of the ambisense S segment recognized by the transcription complex. A mutational analysis of the sequences at the 3' end of the S-like genomic or antigenomic RNA was undertaken. The data indicated that the minimal sequence required for transcription resides in the 13 first 3' nucleotides of the genomic or antigenomic RNA. In these sequences, two regions appeared crucial: the bases at positions 3 to 8 and the purine at position 13. In addition, the terminal repeat ...GU could be deleted without affecting significantly the template activity of the RNA. These data support the prime and realign mechanism proposed recently for Bunya- and Arenaviruses