Current egg-based influenza vaccine production technology, which is labor intensive and slow, would not be able to meet demand during an influenza pandemic. Thus, interest in the emerging technology of using mammalian cells for vaccine production has been great. In this study, Madin-Darby canine kidney (MDCK) cells using microcarrier culture systems were established to produce inactivated whole-virus H5N1 vaccine. The current clade-1 influenza H5N1 vaccine virus (NIBRG-14) was provided by the UK National Institute for Biological Standards and Control. Various process parameters were first optimized in 100-mL scale spinner flasks then scaled up to a 1-L scale bioreactor system. In the 1-L scale bioreactor system, peak virus titer could reach 10(8-9)TCID50/mL using serum-containing medium. After purification and inactivation, hemagglutinin (HA) protein content reached 31.56-43.96 microg/mL in two different runs. In mice immunogenicity studies, two doses of the purified vaccine antigen adjuvanted with aluminum phosphate induced good immune responses in 0.2 and 1.0 microg HA dosages (geometric mean titers of hemagglutination-inhibition antibody: 113 and 242, respectively). This study demonstrates the feasibility of the development of MDCK cell-based inactivated influenza H5 vaccines in microcarrier culture systems and could be valuable to many countries that are planning to establish manufacturing capacity for influenza vaccines.