Acini of human tracheobronchial submucosal glands were isolated by enzymatic disaggregation, and, when plated on flasks coated with human placental collagen (HPC) in media containing Ultroser G serum substitute (USG) and a variety of growth factors (GF), they became confluent after 14-20 days. The cells were then isolated by trypsinization and replated in media containing USG and GF at 10(6) cells/cm2 on porous-bottomed inserts coated with HPC. Confluent monolayers formed on day 1 after replating and were studied on day 10. Transepithelial resistance and short-circuit current (Isc) were 578 +/- 89 omega.cm2 and 12.9 +/- 1.9 microA/cm2 (means +/- SE, n = 23 cell sheets). The potency sequence for stimulation of Isc by mediators was methacholine greater than bradykinin greater than isoproterenol approximately or equal to phenylephrine. Amiloride decreased baseline Isc by 42 +/- 9% (n = 6 cell sheets) but had little effect on the Isc response to mediators. Diphenylamine-2-carboxylic acid, however, had no effect on baseline Isc but markedly inhibited the Isc response to all mediators. These results show that submucosal gland cells from human trachea can be grown in culture to produce epithelial sheets of high resistance, which secrete Cl in response to bradykinin and alpha- and beta-adrenergic and cholinergic agents.