The effect of brassinolide (BR) on cell growth and shikonin and its derivative formation in Onosma paniculatum cell culture was studied. BR addition with IAA and BAP (+BR/+IAA/+BAP) in B(5) medium slightly increased the cell growth at 0.01-0.1 ppb concentration compared with a growth control (-BR/+IAA/+BAP). Only BR addition (+BR/-IAA/-BAP) at 0.001-100 ppb in B(5) medium significantly increased the cell fresh weight compared with a growth control (-BR/-IAA/-BAP). The same concentration of BR tested at 0-1000 ppb increased the cell fresh weight of +IAA/+BAP significantly more than that of -IAA/-BAP. BR at 0.001-0.1 ppb with IAA and BAP added (+BR/+IAA/+BAP) in M(9) medium increased shikonin and its derivative content markedly by 31-87%, compared with its control (-BR/+IAA/+BAP). BR at 0.001-1000 ppb without IAA and BAP added to M(9) medium (+BR/-IAA/-BAP) also increased shikonin and its derivative content compared with its control (-BR/-IAA/-BAP). However, the amount of shikonin and derivative formed of +IAA/+BAP was greater than that of -IAA/-BAP only at the same concentration of BR at 0-1 ppb. These combined results show that BR at 0.01 ppb with IAA and BAP added was the best for cell growth and shikonin formation. Formation of shikonin and its derivative by adding BR at 0.01 ppb with IAA and BAP (+BR/+IAA/+BAP) in M(9) medium was significantly enhanced 4 days after BR addition compared with a production control (-BR/+IAA/+BAP). In contrast, +BR/-IAA/-BAP vs. -BR/-IAA/-BAP was not as effective as +BR/+IAA/+BAP vs. -BR/+IAA/+BAP for the shikonin formation. The time course study for shikonin formation also showed that +BR/+IAA/+BAP and -BP/+IAA/+BAP only slightly increased cell growth in M(9) medium. Similarly, soluble protein content in the cells treated by BR at 0.01 ppb with IAA and BAP (+BR/+IAA/+BAP) exceeded that of the control (-BR/+IAA/+BAP) 4 days after BR addition. And +BR/-IAA/-BAP only slightly increased the soluble protein content over that of -BR/-IAA/-BAP.