In women, during pregnancy, there is decreased motility of the gastrointestinal tract leading to a delay in gastric emptying and an increase in colonic transit time. Whether the rise in estradiol (E₂) or progesterone (P₄) is responsible for this effect is controversial. As the nitrergic component of the nonadrenergic, noncholinergic (NANC) nerves is responsible for modulating gastrointestinal motility in vivo, the purpose of this study was to evaluate whether the increased release of nitric oxide (NO) from the nitrergic component of the NANC nerves innervating the gastric fundus and colon that occurs during late pregnancy in rats is mediated by E₂ or P₄. Ovariectomized rats treated with E₂ or P₄ alone or in combination were used for our studies. We also wanted to assess the cellular and molecular mechanisms involved. The NANC activity was studied by assessing changes in tone after application of electric field stimulation (EFS). The role of NO was determined by observing the effects of EFS in the presence and absence of the NO synthase (NOS) inhibitor N^G-nitro-L-arginine methyl ester (L-NAME) and the reversibility of the effects of L-NAME by L-arginine. Our studies indicated that there was increased magnitude of relaxation of isolated strips of rat gastric fundus and rat colon after application of EFS to tissues obtained from animals treated with E₂ alone or a combination of E₂ + P₄ but not from those treated with P₄ alone. L-NAME attenuated relaxation responses in E₂- and E₂ + P₄-treated animals. To elucidate whether the increased NO release may be due to an increase in neuronal NOS (nNOS) protein, we used both Western blot analysis and immunohistochemistry. We also used RT-PCR to determine whether there was an increase in nNOS mRNA after treatment with sex steroids. In nonpregnant animals, nNOS was detected by Western blot in the fundus and the colon and was barely detectable in the ileum. In pregnancy, there was an increase in nNOS in both the gastric fundus and the colon. The nNOS protein was also increased in ovariectomized animals treated with either E₂ alone or E₂ + P₄ but not P₄ alone when compared with ovariectomized animals receiving vehicle. Our results indicated that there was an increase in nNOS protein that was localized to the neurons of the myenteric plexus in the gastric fundus and colon in E₂- and E₂ + P₄-treated animals, but this increase was not observed in animals treated with P₄ alone. This increase in nNOS protein was accompanied by an increase in nNOS mRNA. These results suggest the possibility that E₂, rather than P₄, may be responsible for the delay in gastric emptying and increase in colonic transit time observed in pregnancy.