Recently, expanded clinical trials using retrovirus-mediated gene therapy require a large volumes of high retroviral vector titers. One approach to achieve high titers of retroviral vector is through the knowledge of the kinetics of retroviral vector production and inactivation in various culture conditions. Effect of serum concentration and temperature on retroviral vector production from producer cells were investigated. Viral vector production at 10% serum concentration was about 10-fold higher than that at 1% serum concentration, because high serum concentration enhanced cell growth and vector production. However, the serum concentration did not significantly influence on the half-lives of retroviral vector, ranging in 2.0-2.7 hours. The culture temperature in the range of 30℃-37℃ also significantly influenced retroviral vector production. Although the m decreased at lower temperature, the half-life of retroviral vector significantly increased. The half-lives of retroviral vector were incubated at 37℃, 34℃, 32℃, and 30℃ was approximately 1.8, 3.6, 5.3, and 9.1 hrs, respectively. Thus, the optimal temperature for retroviral vector production was 34℃. Compared with batch culture, the retroviral vector titers was enhanced in repeated fed-batch culture by 3- to 12-fold. In repeated fed-batch cultivation, the temperature shift from 37℃ in cell growth phase to 34℃ in vector production phase led to 200% increase in viral vector titers compared without temperature shift.