E2 exerts inhibitory actions on nitric oxide pathway in rat adult pituitary glands. Previously we have reported that in vivo E2 acute treatment had opposite effects on sGC subunits, increasing 1 and decreasing 1 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 also are estrogen receptor-dependent. These results suggest that E2 is the main effector of these changes. Since sGC subunit increases while the sGC activity decreases we study if other less active isoforms of sGC subunit are expressed. Here we showed for the first time that the sGC 2 and sGC 2 inhibitory (2i) isoforms are expressed in this gland, but only sGC 2i mRNA increased after E2 acute treatment. Finally, to test if E2 effects take place under a physiological condition, sGC subunits' expression was monitored over estrous cycle. sGC 1, 1 and 2i 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 down-regulates sGC activity in rat anterior pituitary gland.