In the context of infections with highly pathogenic influenza A viruses the PB1-F2 protein contributes to virulence and enhances lung inflammation. In contrast, its role in the pathogenesis of seasonal influenza strains is less clear, especially in the H1N1 subtype, where strains can have a full length 87-90 amino acid protein, a truncated 57 amino acid version, or lack the protein altogether. Towards this, we introduced the full length 1918 PB1-F2, or prevented PB1-F2 expression, in H1N1 A/USSR/90/77, a seasonal strain that naturally expresses a truncated PB1-F2. All viruses replicated with similar efficiency in ferret or macaque ex vivo lung cultures and elicited similar cytokine mRNA profiles. In contrast, the virus expressing the 1918 PB1-F2 protein caused a delay of pro-inflammatory responses in ferret blood-derived macrophages, while the PB1-F2 KO virus resulted in a more rapid response. A similar but less pronounced delay in innate immune activation was also observed in the nasal wash cells of ferrets infected with the 1918 PB1-F2-expressing virus. However, the three viruses did not differ in their virulence or clinical course in ferrets, supporting speculations that PB1-F2 is of limited importance for the pathogenesis of primary viral infection with human seasonal H1N1 viruses.