Isolated uteri from estrogenprimed and spayed rats were allowed to contract isotonically for 45 min in a Krebs- Ringer-bicarbonate-glucose medium gassed with 95% O2:5% CO2. Hormones were then added to the bath and the uteri were assayed for active phosphorylase a and total phosphorylase t. Epinephrine increased phosphorylase a activity 16 % in uteri from spayed rats, 45 % in uteri estrogen-primed for 6 hr and 98% in uteri estrogen-primed for 48 hr. The increase was maximal within 2 min and the activity declined within 10 min. Chronic addition of epinephrine to the bath maintained significantly high levels of phosphorylase a for 10 min. Histamine, 5-hydroxytryptamine and oxytocin also increased uterine phosphorylase a within 2 min. The latter 2 substances produced sustained uterine contractions, whereas histamine and epinephrine caused relaxation. Uteri equilibrated in the medium without glucose had higher phosphorylase a levels than those equilibrated in the standard medium. Epinephrine added to glucose-free medium stimulated uterine phosphorylase a activity to the highest levels obtained in all experiments. Glycogen concentration in uteri equilibrated 45–65 min in the standard medium was significantly lower than that present before equilibration. Stimulation with epinephrine for periods of 2–20 min decreased the uterine glycogen levels slightly below the control levels (5–17%) but in only one instance was the decrease significant. Glycogen levels in uteri equilibrated in glucose-free medium were slightly lower than those obtained in uteri in the standard medium. Although epinephrine was shown previously to decrease phosphorylase a in smooth muscle of the rat uterus when given in vivo, these experiments show that epinephrine increases uterine phosphorylase a activity in vitro as it does in striated muscle and liver, reported by others.