The hormonal requirement for functional differentiation of chick embryo pancreas were investigated by using organ cultures in chemically defined medium. The hormones tested were prednisolone, insulin and thyroxine, and the parameters examined were alpha-amylase (EC 3.2.1.1) and chymotrypsinogen (EC 3.4.4.5) activities, and the ultrastructure of the tissues. Addition of prednisolone alone to explants from 14-day-old chicken embryo pancreas for 3 days increased the activities of amylase and chymotrypsinogen in the tissues by 3.4- and 6.6-fold, respectively, those of tissues before cultivation. Neither thyroxine or insulin alone, nor both hormones together affected pancreatic exocrine differentiation. Thyroxine enhanced the effect of prednisolone on both enzymes, but insulin did not. When the explants were cultured in the medium containing all three hormones, maximum enzyme activities were observed; amylase or chymotrypsinogen activity being 7- or 18-fold, respectively, that of tissues before cultivation. But these three hormones were not simultaneously necessary. Morphological differentiation was also observed in explants cultuvated in medium containing these three hormones. These results suggest that glucocorticoids are essential for normal differentiation of chick pancreas during the late fetal period, possibly with insulin and thyroxine, and also support the idea that pancreatic enzymes are controlled separately.