The development of pancreatic amylase activity was examined in rats fed in regular cages or in special cages, designed so the pups could not reach solid food to prevent weaning. In both groups, the amylase activity in zymogen granules increased in rat pups aged 14 days, peaked at 18 days, and thereafter remained at a 1.6-fold higher level than at 14 days of age. An increase in the plasma concentration of immunoreactive insulin preceded the increase of amylase activity, whereas the plasma concentration of C-peptide, indicating the secretion rate of endogenous insulin, remained unchanged. The administration of insulin at 20 ng/ml (the physiological concentration) in the milk formula caused an increase in the plasma insulin concentration of 17-day-old pups. In addition, increased pancreatic amylase activity was observed in 17-day-old rats raised on milk formula to which insulin was added. We propose that the increase of amylase activity at the beginning of weaning is dependent on the milk-borne insulin and not on the dietary change in rats.