Immature lung development continues to be one of the major complications with preterm birth. Optimal postnatal respiratory function depends on the co-ordinated development of lung branching morphogenesis and fluid secretion in the fetal lung. These processes start during the pseudoglandular stage (weeks 9 – 17 in humans, and embryonic day (E)11.5 – 16.5 in the mouse), and their impairment results in significant and long-lasting morbidity which extends well into adulthood. Previously we have shown that lung development occurs in a relatively hypercalcemic environment (~1.7 mM for the fetus v ~1.2 mM for a normocalcemic adult), and that this relative hypercalcaemia is an important environmental signal for optimal lung development. Specifically, using organ explant cultures in chemically defined, serumless conditions, we have shown that fetal hypercalcaemia suppresses lung branching morphogenesis via activation of the calcium-sensing receptor (CaSR) [1]. In contrast, CaSR activation by hypercalcemic conditions stimulates Cl -driven fluid secretion, resulting in a negative transluminal potential difference (PD), the magnitude of which reflects the rate of fluid secretion [2]. The aim of this work was to identify the mechanisms by which CaSR regulates fluid secretion ex vivo, using mouse and human fetal lung explant cultures and in vivo, by immunohistochemical methods. Our results show that a number of chloride-transporting mechanisms, including bestrophin, TMEM16, the Na-K-2Cl cotransporter (NKCC1) and the cystic fibrosis transmembrane conductance regulator (CFTR) are expressed in both mouse and human fetal lungs. In pseudoglandular fetal lung explants, activation of the CaSR, via either the mimicking fetal hypercalcemia (% PD: 100 ± 16% 1.05 mM Ca2+ control vs 188 ± 31% 1.70 mM Ca2+, p < 0.05, one-way ANOVA, Tukey post) or through the use of the CaSR-specific activator NPS-R568 (%PD: 100% Control vs. 200 ± 45% 100 nM R568, p < 0.05, paired t-test), leads to an increase in fluid secretion. CaSR-mediated activation of fluid secretion was prevented by specific CFTR inhibition using 10 µM CFTR(Inh)-172 (% PD: 188 ± 31% 1.70 mM Ca2+ vs. 71 ± 15% + CFTR(Inh)-172 , p < 0.01, one-way ANOVA, Tuckey post; 100% R568 vs 75 ± 22% R568 + CFTR(Inh)-172, p < 0.05, paired t-test), but was not affected by inhibition of other Ca -activated Cl channels using the anion exchange inhibitor DIDS (100 µM, p > 0.05, one-way ANOVA, Tukey post). Furthermore, CaSR-mediated opening of CFTR requires activation of adenylate cyclase, as shown by using an in vitro high throughput screening model (p < 0.01, one-way ANOVA, Tukey post) [3]. Consistent with these findings, expression of type 1 Ca -activated adenylate cyclase was found in week 10 human fetal lung epithelium. In conclusion, this study indicates that fetal hypercalcaemia, acting thorough the CaSR, regulates lung growth and fluid secretion via adenylate cyclase dependent activation of the CFTR chloride channel.