Cells from the dog's tracheal mucosa formed confluent epithelial sheets in culture. Typical tight junctions separated the apical from the basolateral portion of the cell membrane. The apical portion of the cell contained numerous short microvilli and a pronounced glycocalyx. The basolateral portion of the plasma membrane was unspecialized except for extensive gap junctions between cells. Freeze-fracture showed that the cultured cells lacked the basolateral square (orthogonal) arrays of the original tissue, particles previously implicated in ion transport. Formation of domes indicated the presence of active fluid absorption. Domes appeared between days 4 and 8 of culture and persisted for about 1 week. Cell sheets showed a transepithelial resistance of approximately equal to 400 omega X cm2 and a short-circuit current (Isc) of approximately 5 microA X cm-2. The effects of transport inhibitors indicated that both active Na absorption and active Cl secretion were present. Isc was increased by isoproterenol, prostaglandins E2 and F2 alpha, vasoactive intestinal peptide, dibutyryl cyclic AMP, leukotrienes C4 and D4, and bradykinin. These changes were probably due to stimulation of active Cl secretion.