We previously demonstrated that norepinephrine (NE) induction of cAMP accumulation in slices of the preoptic area (POA) and middle hypothalamus (MH) is reduced by in vivo administration of progesterone to estradiol-primed rats, apparently by eliminating α<sub>1</sub>-receptor augmentation of β-receptor-stimulated cAMP formation. The present studies examined whether in vitro exposure to progesterone would also depress NE-stimulated cAMP synthesis. POA and MH slices from estradiol-primed females were incubated with 20 n M progesterone for 5-30 min prior to addition of 100 µ M NE. Pre-incubation of slices with progesterone for as little as 5 min significantly suppressed NE-stimulated cAMP formation by greater than 60%. This effect was estrogen-dependent in that progesterone in vitro did not inhibit NE-stimulated cAMP accumulation in slices from ovariectomized rats not pretreated with estradiol. Isoproterenol, a β-adrenergic agonist, elevated cAMP to the same extent in slices from estradiol-primed females incubated with and without progesterone in vitro; however, the α<sub>1</sub>-agonist, phenylephrine, was unable to augment cAMP formation in slices incubated in vitro with progesterone for 5 min prior to drug challenge. To determine whether the rapid effects of progesterone may be exerted at the level of the plasma membrane, we employed progesterone conjugated to bovine serum albumin at carbon 3 (P-3-BSA). Slices from estradiol-primed rats incubated with P-3-BSA for 5 min did not exhibit an α<sub>1</sub>-receptor augmentation of β-receptor-stimulated cAMP accumulation. These data indicate that progesterone may have rapid, non-genomic effects on α<sub>1</sub>-adrenergic receptor coupling to second-messenger systems in the hypothalamus of female rats.