A chemically defined protein free medium, DF6S, was developed for the cultivation of a recombinant Chinese hamster ovary cell line (CHO2DS) producing human prothrombin in suspension batch culture. DF6S was formulated by optimizing DME/F12 with amino acids and supplementing the optimized DME/F12 with aurintricarboxylic acid, ethanolamine, ferric sulfate, Pluronic F68, putrescine and sodium pyruvate. From a seeding density of 2.3 x 10(5) cells ml(-1), CHO2DS cells grown in suspension in DF6S medium reached a maximal cell density of 1.92 x 10(6) cells ml(-1) with an accumulated prothrombin concentration of 16.7 mg l(-1) after 6 days in culture.