Spodoptera frugiperda insect cells (IPLB-Sf21-AE) (Sf21), infected with baculovirus expression vectors during their exponential growth phase, are commonly used to produce a variety of heterologous recombinant proteins. In the present study the culture conditions of these insect cells were studied to establish high-density suspension cultures with prolonged exponential growth phases. The Sf21 cells were grown in 125-ml spinner flasks using five different culture media supplemented with 5% fetal calf serum and four protein-free or low-protein culture media. The best results were achieved in EX-CELL 401 (protein-free media) and in IPL-4I modified with 2.5 g l(-1) tryptose phosphate broth (serum-supplemented media), respectively. The latter was used for further batch and continuous cultivation of Sf21 cells in a perfused 1.4-l stirred-tank bioreactor with special attention to the oxygen requirement of these cells. Optimal growth was found at an oxygen concentration of 70% air saturation, resulting in a prolonged exponential growth phase that could be maintained for more than 16 days. A maximum cell density of 5.5 x 10(7) viable cells ml(-1) was achieved.