17beta-estradiol (E2) exerts inhibitory actions on the nitric oxide pathway in rat adult pituitary glands. Previously, we reported that in vivo E2 acute treatment had opposite effects on soluble guanylyl cyclase (sGC) subunits, increasing alpha1- and decreasing beta1-subunit protein and mRNA expression and decreasing sGC activity in immature rats. Here we studied the E2 effect on sGC protein and mRNA expression in anterior pituitary gland from adult female rats to address whether the maturation of the hypothalamus-pituitary axis influences its effects and to corroborate whether these effects occur in physiological conditions such as during estrous cycle. E2 administration causes the same effect on sGC as seen in immature rats, and these effects are estrogen receptor dependent. These results suggest that E2 is the main effector of these changes. Since the sGC alpha-subunit increases while the sGC activity decreases, we studied if other less active isoforms of the sGC alpha-subunit are expressed. Here we show for the first time that sGCalpha2 and sGCalpha2 inhibitory (alpha2i) isoforms are expressed in this gland, but only sGCalpha2i mRNA increased after E2 acute treatment. Finally, to test whether E2 effects take place under a physiological condition, sGC subunit expression was monitored over estrous cycle. sGCalpha1, -beta1, and -alpha2i fluctuate along estrous cycle, and these changes are directly related with E2 level fluctuations rather than to NO level variations. These findings show that E2 physiologically regulates sGC expression and highlight a novel mechanism by which E2 downregulates sGC activity in rat anterior pituitary gland.