It has been shown that both 3' and 5' conserved termini of influenza A virus virion RNA are involved in the initiation of transcription. An RNA-fork model has been proposed, according to which there is a crucial double-stranded region formed by complementary bases at positions 10 to 12 of the 3' terminus and bases at positions 11' to 13' of the 5' terminus, which are extended by 2 or 3 segment-specific base pairs. The two termini at positions 1 to 9 and 1' to 10' in the 3' and 5' termini, respectively, are in a single-stranded conformation. Here we further characterize this model, focusing on the individual roles of the proposed duplex region and the proposed two single-stranded ends. Residues within the conserved 5' terminus that are involved in the initiation of transcription were determined. Single, double, and triple mutations in the proposed duplex region provided further evidence that, for the initiation of transcription in vitro, the duplex RNA is more important than the actual sequence of these residues, although some restrictions in sequence were apparent. On the other hand, there was evidence that base pairing is not required at residues 1 to 7. We propose that the 5' terminus of virion RNA should be treated as an integral part of the virion RNA promoter and discuss a possible mechanism for the recognition of the virion RNA promoter by the influenza A virus RNA polymerase complex.