Aggregate formation of recombinant Chinese hamster ovary (rCHO) cells capable of producing granulocyte colony-stimulating factor (G-CSF), using medium lacking cell adhesion materials in a repeated batch culture, was examined together with cell growth, cell viability and G-CSF production. The rCHO culture was conducted in a rotary shaker and the medium was changed every Eve days. The formation of stable cell aggregates with high reproducibility was observed after the first medium change. The size of the cell aggregates (consisting of several 10s to 40,000 cells) formed during the repeated batch culture ranged from 30 to 600 mu m. The cell density of the aggregates reached as high as 2 x 10(6) cells/ml and the viability was maintained at more than 80% for 19 d. Changing the medium to avoid glucose exhaustion effectively maintained the cell density, cell viability and G-CSF productivity at high levels.