The effects of antioxidants on the apoptosis of Chinese hamster ovary (CHO) 1-15,500 cells and production of tissue plasminogen activator (tPA) in suspension culture were investigated. After cell growth to 2 x 10(5) cells/ml in Ham's F12 medium containing 10% newborn bovine serum (NBS) in a spinner bottle, CHO cells were maintained for 6 d in Ham's F12 medium containing 0 or 0.4% NBS and 10 mM antioxidants, namely, L-ascorbic acid 2-phosphate (VCP) or the reduced form of glutathione (GSH). The viable cell concentrations at day 6 in the serum-free culture with GSH and in the low-serum culture with VCP or GSH were 0.57, 1.04 And 1.69 x 10(5) cells/ml, respectively, while those in the serum-free and low-serum cultures without the antioxidants were only 2.33 and 1.17 x 10(3) cells/ml, respectively. The percentages of apoptotic cells in the serum-free and low-serum cultures with VCP (73.2, 44.6%) and GSH (76.9, 38.6%) measured using a flowcytometer after annexinV-FITC/propidium iodide staining were markedly lower than those in the cultures without antioxidants (96.3, 92.5%). The percentage of cells having a high mitochondrial membrane potential among the viable cells in the cultures with antioxidants determined using a flowcytometer after 5,5 ' ,6,6-tetrachloro-1,1 ' ,3,3 ' -tetraethylbenzimidazolylcarbocyanine iodide staining was clearly higher than those in the cultures without the antioxidants. The production of tPA in the serum-free and low-serum media with VCP (0.282, 2.02 mg/l) or GSH (1.01, 1.61 mg/l) was markedly higher than that in the cultures without the antioxidants (0.275, 0.689 mg/l). Consequently, the suppression of apoptosis through the maintenance of the membrane potential of mitochondria by VCP or GSH resulted in a marked increase in tPA production by CHO cells in the serum-free and low-serum cultures.