A final step in the influenza virus replication cycle is the assembly of the viral structural proteins and packaging of the eight segments of vRNA into a fully infectious virion. The process by which the RNA genome is packaged efficiently remains poorly understood. In an approach to analyze how vRNA is packaged, we rescued a seven-segmented virus, lacking the hemagglutinin (HA) vRNA (deltaHA virus). This virus could be passaged in cells constitutively expressing HA protein, but it was attenuated in comparison to wt A/WSN/33 virus. Supplementing the deltaHA virus with an artificial segment containing GFP or RFP with HA packaging regions (forty-five 3´ and eighty 5´ nucleotides) partially restored the growth of this virus to wild-type levels. The absence of the HA vRNA in the deltaHA virus resulted in a 40-60% reduction in packaging of the PA, NP, NA, M and NS vRNAs as measured by qPCR and packaging of these vRNAs was partially restored in the presence of GFP/RFP packaging constructs. To further define nucleotides of the HA coding sequence which are important for vRNA packaging, synonymous mutations were introduced into the full length HA cDNA of influenza A/WSN/33 and A/Puerto Rico/8/34 viruses and mutant viruses were rescued. qPCR analysis of vRNAs packaged in these mutant viruses identified a key region of the open reading frame (nucleotides 1659-1671) that is critical for efficient packaging of an influenza virus H1 HA segment.