Background The RNA-dependent RNA polymerase of Influenza A virus is a determinant of viral pathogenicity and host range that is responsible for transcribing and replicating the negative sense segmented viral genome (vRNA). Transcription produces capped and polyadenylated mRNAs whereas genome replication involves the synthesis of an alternative plus-sense transcript (cRNA) with unmodified termini that is copied back to vRNA. Viral mRNA transcription predominates at early stages of viral infection, while later, negative sense genome replication is favoured. However, the "switch" that regulates the transition from transcription to replication is poorly understood. Results We show that temperature strongly affects the balance between plus and minus-sense RNA synthesis with high temperature causing a large decrease in vRNA accumulation, a moderate decrease in cRNA levels but (depending on genome segment) either increased or unchanged levels of mRNA. We found no evidence implicating cellular heat shock protein activity in this effect despite the known association of hsp70 and hsp90 with viral polymerase components. Temperature-shift experiments indicated that polymerase synthesised at 41°C maintained transcriptional activity even though genome replication failed. Reduced polymerase association with viral RNA was seen in vivo and in confirmation of this, in vitro binding assays showed that temperature increased the rate of dissociation of polymerase from both positive and negative sense promoters. However, the interaction of polymerase with the cRNA promoter was particularly heat labile, showing rapid dissociation even at 37°C. This suggested that vRNA synthesis fails at elevated temperatures because the polymerase does not bind the promoter. In support of this hypothesis, a mutant cRNA promoter with vRNA-like sequence elements supported vRNA synthesis at higher temperatures than the wild-type promoter. Conclusion The differential stability of negative and positive sense polymerase-promoter complexes explains why high temperature favours transcription over replication and has implications for the control of viral RNA synthesis at physiological temperatures. Furthermore, given the different body temperatures of birds and man, these finding suggest molecular hypotheses for how polymerase function may affect host range.