Defining the effects of neuraminidase inhibitors on influenza virus infection may provide important information for the treatment of patients. The effects of neuraminidase inhibitors have been examined using various methods, including viral release from kidney cells. However, the effects of neuraminidase inhibitors on viral release from primary cultures of human tracheal epithelial cells, which retain functions of the original tissues, have not been studied. The effects of neuraminidase inhibitors on the replication of the pandemic influenza virus [A/Sendai-H/N0633/2009 (H1N1) pdm09] and the seasonal influenza virus [A/Sendai-H/216/2009 (H1N1)] that was isolated during the 2008–2009 season were examined. The virus stocks were generated by infecting tracheal cells with the pandemic or seasonal influenza virus. Four types of inhibitors (oseltamivir, zanamivir, laninamivir, and peramivir) reduced pandemic viral titers and concentrations of the cytokines interleukin-6 and tumor necrosis factor-α in supernatants and viral RNA in cells. However, oseltamivir did not reduce seasonal viral titers, cytokine concentrations and viral RNA, and the 50% inhibitory concentration (IC50) of oseltamivir for neuraminidase activity in the seasonal virus was 300-fold higher than that observed for the pandemic influenza virus. The seasonal influenza virus had an oseltamivir-resistant genotype. The magnitude of the IC50 values of the neuraminidase inhibitors for the seasonal influenza virus was inversely related to the magnitude of the inhibitory effects on viral release. These methods for measuring the release of virus and inflammatory cytokines from primary cultures of human tracheal epithelium may provide useful information regarding the effects of neuraminidase inhibitors on influenza viruses. J. Med. Virol. 87: 25–34, 2015. © 2014 Wiley Periodicals, Inc.