Because functional changes of the pancreas during the ovarian cycle are not fully understood, effects of gonadal steroid hormones on pancreatic amylase content and secretion were investigated. The estrus cycle of female rats was confirmed by vaginal smear. To mimic the estrus or the diestrus, estradiol 17beta (25 microg/kg) or progesterone (50 mg/kg) was subcutaneously injected to ovariectomized rats once daily for 2 days. Amylase secretion of pancreatic lobules (approximately 6 mg wet weight) was induced by acetylcholine (10-8 approximately 10-4 M). In normal rats, pancreatic amylase content was not altered during the estrus cycle, whereas spontaneous amylase secretion of pancreatic lobules at the diestrus was significantly higher than that at the estrus. In ovariectomized rat, pancreatic amylase content was markedly reduced, which was restored by either estradiol 17beta or progesterone. Pancreatic lobules of ovariectomized rats spontaneously secreted amylase at the level identical to that at the estrus, which was elevated to the level at the diestrus by progesterone, but not affected by estradiol 17beta. In normal rats, acetylcholine induced amylase secretion much higher at the diestrus than at the estrus. In ovariectomized rats, the acetylcholine-induced amylase secretion was similar to that at the estrus, which was elevated by progesterone, but not affected by estradiol 17beta. We conclude from the above results that both estradiol 17beta and progesterone are necessary to maintain amylase content in the rat pancreas, but only progesterone exerts a stimulatory effect on spontaneous and stimulated amylase secretion in pancreatic lobules of rats.