Analogues of the mRNA 5'-terminal methyl cap structure were found to stimulate the influenza virion RNA-dependent RNA polymerase. The single nucleotide analogue m7GMP was incorporated into RNA during transcription in vitro, and the stimulatory effect was not additive with the primer ApG, suggesting that m7GMP stimulates the virion polymerase by priming virus-specific mRNA synthesis, as has been shown for ApG. By contrast, stimulation by m7G(5')ppp(5')m6AM2-O was additive with that by ApG, and we could not demonstrate incorporation of the similar analogue m7G(5')ppp(5')Am2-O into RNA during transcription. We propose that these dinucleotide cap analogues stimulate the virion polymerase by allosteric modulation, independent of priming. This stimulation can be abolished by mutation, without loss of other activities associated with the cap-dependent endonuclease.