Electrophysiological studies of human bronchial epithelial cells in vitro are limited by the scarcity of biological material available for primary culture. To overcome this problem, we set up a protocol in which the cell number is first enlarged in LHC9/RPMI 1640 serum-free medium for up to six passages, each passage giving a four- to eightfold amplification. The cells are then plated at high density on permeable supports. Cell differentiation, monitored by measuring transepithelial potential difference (PD) and electrical resistance (R), is induced with a medium containing serum and a cocktail of different supplements and hormones. Maximal values of PD and R, obtained after 4-7 d of culture on permeable supports, are around -50 mV and 3000-4000 omega/cm2, respectively. Ussing chamber experiments show that basal short-circuit current (Isc) is partially inhibited by the epithelial Na+ channel blocker amiloride. Stimulation with a cAMP-elevating agent induces a Isc increase that is inhibited by the cystic fibrosis transmembrane conductance regulator (CFTR) blocker glibenclamide. Our culture protocol provides a large number of differentiated bronchial epithelial cell monolayers starting from a low amount of material. This characteristic is useful for in vitro studies of ion transport in airway epithelium.