The production of tropane alkaloids during differentiation and de-differentiation of Scopolia parviflora calli was studied. Tropane alkaloid production drastically decreased during calli de-differentiation. Scopolamine (1) production decreased after 10 days of culture, whereas that of hyoscyamine (2) decreased during de-differentiation of root to calli. The production of 1 was enhanced in calli undergoing differentiation to shoot after 60 days of culture, reaching a maximum by 80 days. However, production of hyoscyamine in regenerated plants was lower. The expression level of hyoscyamine 6beta-hydroxylase (H6H), a key biosynthetic enzyme for tropane alkaloids, was significantly increased in 4-week-old calli. This study suggests that the biosynthesis of tropane alkaloids is regulated inversely in de-differentiating Scopolia parviflora calli.