A perfusion system based on a 4-L stirred tank bioreactor and a custom-designed tangential (cross-flow) filter was assembled to realize a scaleup of the Baculovirus Expression Vector System (BEVS). When perfused with 1 to 1.5 vol/day, Spodoptera frugiperda (Sf-9) insect cell cultures grew from 4 x 10(6) to 15 x 10(6) cells/ml over 3 to 4 days. The possibility of maintaining high specific production of recombinant VP6 protein (from bovine rotavirus) after baculovirus infection of the high-density cultures was then assessed. The process consisted of a growth phase in TNMFH + 10% FBS, followed by infection with Bac-BRV6L recombinant baculovirus and a shift to a low-serum (0 to 1%) medium for perfusion during the production phase. Multiple runs were executed, each including a battery of shaker flask controls at various cell densities and serum concentrations. On average, specific rVPG production in the bioreactor amounted to 76% of that found in 20-mL shaker cultures simulating the bioreactor’s high cell density, low serum concentration, and medium renewal rate. Mechanical stress generated by cell/medium separation in the perfusion process reduced cell growth rate but had minimal effect on rVPG production. Our results also indicated that serum concentration during the infection phase affected the rVPG specific production in a cell density-dependent fashion. Although the feasibility of the cell density scale up was demonstrated, optimization is still needed to achieve a truly cost-effective process.