AbstractThe airway epithelial cell line, 16HBE14o−, is an important cell model for studying airway disease. 16HBE14o−cells were originally generated from primary human bronchial epithelial cells by SV40‐mediated immortalization, a process that is associated with genomic instability through long‐term culture. Here, we explore the heterogeneity of these cells, with respect to expression of the cystic fibrosis transmembrane conductance regulator (CFTR) transcript and protein. We isolate clones of 16HBE14o−with stably higher and lower levels of CFTR in comparison to bulk 16HBE14o−, designated CFTRhighand CFTRlow. Detailed characterization of theCFTRlocus in these clones by ATAC‐seq and 4C‐seq showed open chromatin profiles and higher order chromatin structure that correlate withCFTRexpression levels. Transcriptomic profiling of CFTRhighand CFTRlowcells showed that the CFTRhighcells had an elevated inflammatory/innate immune response phenotype. These results encourage caution in interpreting functional data from clonal lines of 16HBE14o−cells, generated after genomic or other manipulations.