Ionocytes are a new type of rare airway epithelial cells expressing Ascl3 and Foxi1 transcription factors, as well as 50% of Cftr-transcripts in mouse airways. However, their role and precise location remain mostly unexplored. This research aimed to shed light on ionocyte ontogeny and distribution within the mouse airway epithelium. Mice were bred in the C57Bl6/J background and maintained in the Specific Pathogen Free mouse facility of Centro de Estudios Científicos (CECs) with access to food and water ad libitum. We used 1,7, 10, 14,21 and 28-days-old wild-type mice, 6 and 8-week-old, and 1 year wild-type and 14-days-old 6 and 8-week-old CftrΔF508/ΔF508 animals. The tracheas were sliced in frontal sections, and the ionocytes were identified by immunofluorescence against the FOXI1 transcription factor. All values were expressed as mean ± S.E.M. All animal procedures were approved by the institutional IACUC (CECS-2022-03). We found that FOXI1+cells had a triangular shape with a basolateral process. Unexpectedly, we observed that FOXI1+cells appear postnatally close to the submucosal glands (SMG), and its number increases drastically between 6 and 8-week-old wild type mice. The quantity of FOXI1+cells cells in the adult trachea decreased towards the distal part (proximal=11.2±1.2 vs distal=4.0±0.9 FOXI1+cells/mm basal lamina, n=7, P=<0,001, t-test), and we did not find significative differences between WT and CftrΔF508/ΔF508 adult mouse. FOXI1+cells were often observed in the epithelia around the collecting duct exit and in the collecting duct epithelium of the SMG. In conclusion, our study provides new insights into the localization and distribution of ionocytes in the mouse airway epithelium. Our results indicate that ionocytes may play a role in regulating mucus composition in upper airways. We suggest that age-dependent changes in cell quantity might reflect the need of increased CFTR function in adult stages. Further research is needed to fully understand the function of ionocytes in the airway and their potential role in respiratory diseases such as cystic fibrosis. Proyecto Postdoctoral 3220672 Proyecto FONDECYT 1221257. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.