Previous studies showed that 17-estradiol (17-E2) regulates the nitric oxide (NO)/soluble guanylyl cyclase (sGC)/cGMP pathway in many tissues. Evidence from our laboratory indicates that 17-E2 disrupts the inhibitory effect of NO on prolactin release, decreasing sGC activity and affecting the cGMP pathway in anterior pituitary gland of adult ovariectomized and estrogenized rats. To ascertain the mechanisms by which 17-E2 affects sGC activity we investigated the in vivo and in vitro effects of 17-E2 on sGC protein and mRNA expression in anterior pituitary gland from immature female rats. In the present work, we showed that 17-E2 acute treatment exerted opposite effects on the two sGC subunits, increasing 1 and decreasing 1 subunit protein and mRNA expression. This action on sGC protein expression was maximal 6 to 9 h post 17-E2 administration. 17-E2 also caused the same effect on mRNA expression at earlier times. Concomitantly, 17-E2 dramatically decreased sGC activity 6 and 9 h after injection. These effects were specific of 17-E2, since they were not observed with the administration of other steroids such as progesterone and 17-estradiol. This inhibitory action of 17-E2 on sGC also required the activation of estrogen receptor (ER), since treatment with the pure ER antagonist ICI 182,780 completely blocked 17-E2 action. 17-E2 acute treatment caused the same effects on pituitary cells in culture. These results suggest that 17-E2 exerts an acute inhibitory effect on sGC in anterior pituitary gland by down-regulating sGC 1 subunit and sGC activity in a specific, ER-dependent manner.