Submucosal glands are a major site of cystic fibrosis transmembrane conductance regulator (CFTR) expression in the human airway and may play an important role in the pathogenesis of cystic fibrosis. In humans, strategies for gene targeting to submucosal glands will likely be dependent on in utero gene transfer to submucosal gland progenitors because of the inaccessibility of these regions from the fully developed airway. However, little is known about the ontogeny of CFTR gene expression in developing submucosal glands and the potential functional role(s) CFTR may have during gland development. To this end, we describe the partial cloning of the ferret CFTR cDNA which was used for in situ mRNA localization studies in developing ferret trachea. The ferret animal model is attractive for studies pertaining to the development of tracheal submucosal glands because postnatal tracheal development in this species mirrors in utero gland development in humans. Sequence analysis of the first nucleotide binding domain (NBD1) from ferret CFTR revealed a striking homology to the human gene at both the DNA (94.4%) and amino acid level (97%). Interestingly, this high level of amino acid homology extends to a group of mammalian species (ferret, human, sheep, and bovine) which have similar lung morphologies with respect to the presence of surface airway goblet cells and submucosal glands. In contrast, mouse and rat airways which are quite divergent from those of ferret, human, sheep, and bovine species with respect to secretory cell types in the airway and abundance of submucosal glands, demonstrate significantly less homology (80%) with respect to CFTR amino acid sequence in the NBD1 domain. Given that traditional phylogenetic classification of these species does not mirror the evolutionary conservation of CFTR, such findings would suggest that certain aspects of lung morphology may be reflected in the evolutionary conservation of CFTR NBD1 amino acid sequences. CFTR in situ hybridization studies in ferret trachea demonstrate a developmental increase (3-4 fold) in CFTR mRNA expression within the surface airway epithelium between -2 day to 5 wk which mirrors an increase in ciliogenesis over this time frame. Additionally, all stages of gland development including the most primordial gland-forming buds could be seen to contain infrequent cells which highly express CFTR at a level that remained constant throughout development. Such findings suggest that differentiation of gland progenitor cells to CFTR-expressing submucosal gland cells occurs very early within submucosal gland development and morphogenesis.