To develop serum-free (SF) media for the production of humanized antibody from recombinant Chinese hamster ovary (rCHO cells, a statistical optimization approach based on a Plackett-Burman design was adopted. A basal medium was prepared by supplementing alpha-minimal essential medium (alpha-MEM) with Fe(NO3)(3). 9H(2)O, CuCl2, ZnSO4. 7H(2)O, and Na2SeO3 which are generally contained in SF medium formulations. Insulin, transferrin, and ethanolamine were also supplemented to the basal medium to determine their optimal concentrations. From this statistical analysis, serine, phenylalanine, and tyrosine were identified as important determinants for cell growth. Also, putrescine, linoleic acid, and hydrocortisone were shown to be important for both cell growth and antibody production. The SF medium was formulated by supplementing the basal medium with components showing positive effects on cell growth and/or antibody production. Cell growth and antibody production in this SF medium were comparable to those in alpha-MEM supplemented with 5% dialyzed fetal bovine serum. Taken together, the results obtained here show that a Plackett-Burman design facilitates the development of SF media for rCHO cells aimed at producing a humanized antibody.