Recombinant adeno-associated virus (rAAV) is one of the most promising vectors for human gene therapy. However, the production systems that are currently available have a limited capacity and cannot provide sufficient quantities of rAAV for preclinical or clinical trials. Many novel methods for improving rAAV production have been developed, but few researchers have focused on the culture process. In this study, we use a fed-batch culture system to enhance rAAV yield in the baculovirus/insect cell system. When the insect cells were co-infected with MOI = 5 of Bac-GFP at a ratio of 1:9:9 (Bac-GFP: Bac-Rep: Bac-VP), the fed-batch culture achieved optimal rAAV yields. In batch culture, the optimal cell density for producing rAAV was found to be l x 10(6) cells/ml, and the highest rAAV yield (1.22 x 10(8) IVP/ml, 122 IVP/cell) occurred at day 5 post-infection. In the fed-batch culture, rAAV yield reached 2.13 x 10(8) IVP/ml at day 4 post-infection, and the highest rAAV yield was 2.40 x 10(8) IVP/ml (240 IVP/cell) at day 5 post-infection. The cost of the batch and fed-batch cultures is similar; however, the rAAV yield was 2.6-fold higher in the fed-batch culture system compared with that in the batch culture system. Therefore, here we demonstrated an economical and efficient strategy for rAAV production. (C) 2010, The Society for Biotechnology, Japan. All rights reserved.