The effect of ovarian hormones on in vivo gluconeogenesis and glycogen deposition in liver, uterus, skeletal and cardiac muscle was studied. Ovariectomized adult female mice were treated with replacement doses of estradiol, progesterone, both hormones combined, or vehicle only for 15 weeks. Compared with intact control mice, ovariectomy increased gluconeogenesis and reduced the glycogen content of all tissues examined. Treatment with estradiol and progesterone, individually and in combination, increased tissue glycogen deposition. Estradiol alone consistently produced the greatest effect, except on hepatic glycogen, which was maximally increased by the combined estradiol-progesterone regimen. Estradiol markedly reduced gluconeogenesis, and this effect was antagonized by progesterone. The results indicate that the lower plasma glucose concentrations produced by ovarian steroids result in part from reduced glucose neoformation and greater storage of glycogen in liver and muscle tissues.