The chloride permeability of airway and sweat ductal epithelium of cystic fibrosis (CF) patients is decreased. This abnormality could represent an intrinsic characteristic of the epithelial cell or the response to a tonic extrinsic stimulus, in vivo. We cultured airway epithelial cells derived from CF and non-CF individuals under identical conditions that were free from donor-specific factors. Differences in the characteristics of cells that multiplied under these circumstances are unlikely to reflect the effects of extrinsic modulation present in the host. After 8-12 days in culture, the cells of CF and non-CF patients were similar in morphology and intracellular electrolyte content, but the CF cultures took up chloride at a reduced rate. The difference could not be attributed to a higher intracellular potential in CF cells or to the presence of a stilbene anion-sensitive chloride-chloride exchange in non-CF cells. We conclude that epithelial cells from CF patients grown in the absence of extracellular factors of the host express reduced cellular chloride permeability, a defect similar to that found in vivo and in freshly excised nasal epithelium.