In preparation for birth, lung epithelia must switch from net fluid secretion, required for lung development, to net absorption which prepares the lungs for postnatal gas exchange. The apical membrane amiloride-sensitive epithelial Na channel (ENaC) is the rate-limiting step for Na⁺ and fluid absorption. Expression of -ENaC mRNA has been detected in human lung as early as the embryonic stage of development. However, humans express multiple transcripts for -ENaC, containing differing 5'-untranslated regions (UTR) with unknown effects on protein translation, and different ontogenies for individual transcripts could provide a novel mechanism for developmental regulation of ENaC function. To assess the relative expression of the two most abundant -ENaC transcripts (ENaC1 and ENaC2) during lung development, we performed non-radioactive in situ hybridization using probes specific to the alternative 5'-UTRs. Both transcripts were expressed throughout intrauterine lung development (8 to 40 wk gestation), and expression was localized to the surface epithelial cells of the conductive and respiratory airways in both ciliated cells and nonciliated Clara cells. -ENaC mRNA expression was also identified in the serous cells of the submucosal glands surrounding the proximal airways. In the mature prenatal lung, subsets of alveolar Type II (ATII) cells expressed one or both of the ENaC transcripts. Our observations demonstrate that a developmentally regulated switch between -ENaC 5'UTR variants is not the trigger by which the developing human lung becomes a fluid-absorbing organ at birth, that individual ATII cells express neither, one or both of the -ENaC transcripts, and the overall expression is linked to epithelial cell differentiation and lung maturation.