We have developed a technique for expanding primary cultures of human tracheal epithelium while minimizing loss of differentiated structure and function. Cells were seeded at 2 x 10(4) cells/cm2 into T75 flasks and trypsinized when approximately 80% confluent. The dispersed cells were then passaged at the same plating density into further T75 flasks or seeded at 5 x 10(5) cells/cm2 on porous-bottomed inserts and maintained with an air-interface. Differentiation of cells on inserts was assessed from transepithelial electrical resistance (an index of tight junction formation), short-circuit current (an index of transepithelial salt transport), cell numbers, total cell protein, and histology. Unpassaged cells (P0) and cells passaged once (P1) took about a week to become 80% confluent on T75 flasks, with 10-fold and 5-fold increases in cell numbers, respectively. Confluence was achieved in approximately 3 days following plating to inserts. Functionally and structurally, P1 and P2 cells (cells passaged twice) were little different from P0 cells. Thus, within a little over 2 weeks, the numbers of confluent cell sheets can be increased 50-fold with minimal change in function. However, there was a marked decline in differentiation by cells passaged three times (P3), and not all cell preparations could be taken to P4 (cells passaged four times).