Key points: Biochemical and biophysical characterizations of three nonsense mutations of cystic fibrosis transmembrane conductance regulator (CFTR) associated with a severe form of cystic fibrosis (CF) reveal the importance and heterogenous effects of the position of the premature termination codon (PTC) on the CFTR protein function. Electrophysiological studies of W1282X-CFTR, whose PTC is closer to the C-terminus of CFTR, suggest the presence of both C-terminus truncated CFTR proteins that are poorly functional and read-through, full-length products. For G542X- and E60X-CFTR, the only mechanism capable of generating functional proteins is the read-through, but the outcome of read-through products is highly variable depending on the interplay between the missense mutation caused by the read-through and the structural context of the protein. Pharmacological studies of these three PTCs with various CFTR modulators suggest position-dependent therapeutic strategies for these disease-inflicting mutations. Abstract: About one-third of genetic diseases and cancers are caused by the introduction of premature termination codons (PTCs). In theory, the location of the PTC in a gene determines the alternative mechanisms of translation, including premature cessation or reinitiation of translation, and read-through, resulting in differential effects on protein integrity. In this study, we used CFTR as a model system to investigate the positional effect of the PTC because of its well-understood structure-function relationship and pathophysiology. The characterization of three PTC mutations, E60X-, G542X- and W1282X-CFTR revealed heterogenous effects of these PTCs on CFTR function. The W1282X mutation results in both C-terminus truncated and read-through proteins that are partially or fully functional. In contrast, only the read-through protein is functional with E60X- and G542X-CFTR, although abundant N-terminus truncated proteins due to reinitiation of translation were detected in E60X-CFTR. Single-channel studies of the read-through proteins of E60X- and G542X-CFTR demonstrated that both mutations have a single-channel amplitude similar to wild type (WT), and good responses to high-affinity ATP analogues, suggesting intact ion permeation pathways and nucleotide binding domains (NBDs), albeit with reduced open probability (Po). The comparison of the Po of these mutations with the proposed missense mutations revealed potential identities of the read-through products. Importantly, a majority of the functional protein studied responds to CFTR modulators like GLPG1837 and Lumacaftor. These results not only expand current understanding of the molecular (patho)physiology of CFTR, but also infer therapeutic strategies for different PTC mutations at large.