Intracellular cyclic AMP (cAMP) regulates many critical differentiated functions of tracheal epithelial cells. An in vitro model system for reliable study of cAMP metabolism in these cells has been developed. Viable tracheal epithelial cells could be recovered from greater than 50% of necropsy specimens. Culture success rate was not significantly affected by age of subject, endotracheal intubation, or time between death and autopsy, although most specimens were obtained within 24 h of death. Human tracheal epithelial cells grown in primary culture displayed a typical histologic epithelial appearance, and the ultrastructure showed microvilli, junctional complexes, and tonofilaments. The cells uniformly stained with fluorescent antibody to cytokeratin, and expressed receptors for isoproterenol and vasoactive intestinal peptide. Human tracheal epithelial cells grown serum-free in an equal volume mix of Ham's F12 medium and Dulbecco's minimal essential medium containing growth supplements (Medium A) and cholera toxin (CT) had higher basal cAMP levels and greater increase in intracellular cAMP in response to phosphodiesterase inhibition than cells grown in Medium A without CT. Cells grown in Medium A without CT had similar morphology and grew at a comparable rate but attached to the culture substratum less readily than cells grown in Medium A with CT. Cells grown in Medium A without CT had less cAMP response to phosphodiesterase inhibition, less rapid accumulation of cAMP, and greater proportional response to receptor-mediated stimulation of cAMP production compared to cells grown with CT, though the final cAMP levels achieved were comparable.(ABSTRACT TRUNCATED AT 250 WORDS)