The effects of prolactin (PRL) on circulating levels of glucose and insulin, and of estradiol on hyperprolactinemia-induced glucose intolerance of tissues were studied in pituitary-grafted SHN mice (PG mice) and sham-operated controls. Pituitary grafting (PG) decreased blood glucose levels in male mice at 1 and 3 months after the operation but did not alter those in females. PG had little effect on serum insulin levels in males, but increased those in females. In female mice at 2 months after PG, blood glucose levels were significantly higher at 1, 2, and 4 hr after glucose load when compared with those in controls. In contrast, there was no significant difference in blood glucose levels after glucose load between male PG and control mice. The rate at which blood glucose levels decreased was slower in female PG mice than in controls during the 30 min after insulin injection, whereas there was no difference in the rate after insulin injection between male PG and control mice. In ovariectomized (Ovx) mice, no significant difference was found in the blood glucose levels after a glucose load between PG and control groups at 2 months after PG. In Ovx mice treated daily with estrogen, however, a PG-dependent high level of blood glucose was observed after glucose load. These results suggest that hyperprolactinemia decreases glucose tolerance via an increase in insulin resistance in female SHN mice and that estrogen is essential to the expression of the PRL effect.