Primary tracheal epithelial cells obtained from two fetuses with cystic fibrosis (CF) were successfully transfected with a plasmid vector recombined with the large T oncogene of SV40. The resulting tracheal cells were propagated in culture for up to 25 passages and retained the mutations of the CF genes carried by the two fetuses, one heterozygous for the S549N and N1303K substitutions (CFT-1 cells), and the other homozygous for the most common deletion delta F508 (CFT-2 cells). The transfected cells: (a) expressed the SV40 large T oncogene, as determined by immunofluorescence and Northern blot analysis; (b) retained typical epithelial morphology, as assessed by the presence of microvilli, desmosomes, gap junctions, and cytokeratin expression; (c) were fully responsive to the cAMP-stimulating agents isoproterenol, forskolin and vasoactive intestinal peptide for cAMP production and PKA activation; (d) do not produce any tumour in the athymic nude mice; (e) were diploid and tetraploid with a normal chromosomal complement at early passages, and (f) exhibited the abnormal regulation of chloride conductance characteristic of CF. These results indicate that CFT-1 and CFT-2 cells constitute a suitable model for: (a) comparison of the maturation and function of the CFTR protein mutated in the two nucleotide-binding domains; (2) analysis of the biochemical defect in CF epithelial airway cells, (c) development of new therapeutic agents, and correction of the CF defect by gene replacement therapy in vitro.