Canine influenza virus (CIV) is an important emerging pathogen that causes highly contagious respiratory disease in dogs. Secondary bacterial pneumonia is a leading complication of CIV infection. Initial characterizations of CIV-induced respiratory disease suggested alveolar macrophages may be susceptible to virus infection. Studies using other influenza viruses have revealed that alveolar macrophages may play an important role in influenza pathogenesis, and that prior infection of alveolar macrophages with influenza virus augments the cytokine response to bacterial products. The hypothesis for these studies was that CIV induces severe respiratory disease in dogs by infecting pulmonary macrophages and inducing high levels of pro-inflammatory cytokines such as TNF-α and that CIV infection of macrophages induces dysregulated cytokine responses in macrophages when they are subsequently exposed to bacterial pathogens. Infection of alveolar macrophages with CIV was demonstrated by production of virus in macrophage cultures, expression of virus matrix mRNA, and expression of virus antigen in inoculated macrophages. Infection of alveolar macrophages with CIV led to cell death via both necrosis and apoptosis. Following inoculation with CIV, alveolar macrophages produced TNF-α to a similar extent as to when exposed to lipopolysaccharide. This TNF-α production was host-strain specific as inoculation with alveolar macrophages with a genetically related equine influenza virus yielded similar virus matrix mRNA expression yet significantly (p<0.05) less TNF-α production. Prior infection of alveolar macrophages primed them for an exaggerated TNF-α response to lipopolysaccharide, but not to other bacterial products such as lipoteichoic acid, flagellin or unmethylated CpG DNA. This effect was mediated by a much larger accumulation of TNF-α mRNA following LPS exposure in CIV-infected macrophages than in macrophages exposed only to LPS or virus. The mechanism for the increased mRNA remains poorly defined and does not appear to be explained by altered mRNA degradation alone. In conclusion, CIV infects alveolar macrophages and induces TNF-α expression and cell death. Prior infection of alveolar macrophages with CIV augments the TNF-α response to LPS, and this effect is mediated at least in part by increased amounts of TNF-α mRNA.