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To better understand the role of atrial natriuretic peptide (ANP) in the central regulation of hydro-mineral homeostasis, we analysed its expression in rat hypothalamic neurones during gestation and postpartum. These physiological events are characterized by opposing body fluid regulations. Quantitative in situ hybridization analysis showed that starting from mid-pregnancy, ANP mRNA declined in neurones of the preoptic area, periventricular area, lateral hypothalamus and endorhinal nucleus, and remained low at postpartum. By contrast, magnocellular cells in the supraoptic nucleus (SON) showed four- and 10-fold more ANP mRNA in sections from preterm and postpartum rats, respectively, compared to nonpregnant controls (P < 0.001). Oxytocin mRNA paralleled ANP mRNA expression in the SON, whereas vasopressin mRNA rose in early pregnancy and declined thereafter. High hypothalamic ANP concentration at day 21 of gestation versus nonpregnant rats (3.1 ± 0.5 versus 1.8 ± 0.4 ng/mg protein, P < 0.05) suggested that ANP transcript accumulation in the SON is associated with increased utilization of the peptide. The elevation of hypothalamic ANP (two-fold) and ANP receptors by treatment of ovariectomized rats with 17β-oestradiol (25 μg/rat, 10 days) was abolished by coadministration of progesterone. Thus, we concluded that elevated oestradiol at term stimulates ANP synthesis and paracrine ANP activation in the hypothalamus. Overall, we provide experimental, anatomical and molecular evidence for ANP regulation in hypothalamic neurones at preterm and after 17β-oestradiol stimulation. Our study supports the concept that ANP expressed in the SON acts as a peptidergic neurotransmitter involved in water and salt regulation during pregnancy and postpartum.