The effects of the cultivation media, plant growth regulators and inoculum size on the cell growth and 20-hydroxyecdysone production in suspension cultures of Vitex glabrata R.Br. were investigated. The cell growth and 20-hydroxyecdysone formation reach the highest when cells are cultured in the Gamborg's B5 medium supplemented with 2.0mg/L BAP (6-benzylaminopurine) and 1.0mg/L 2,4-D. The maximum 20-hydroxyecdysone productivity, of about 1.1mg/L/day, was observed in the culture with 20% PCV (packed cell volume) of inoculum size. These data also show that the increment of the inoculum size to 20% PCV could increase the productivity in 7-folds.