In this study, we have developed an air-interface culture system in which guinea pig tracheobronchial epithelial (GPTE) cells rapidly form tight monolayers. Enzymatically isolated GPTE cells were plated on collagen-treated polycarbonate microporous cell culture inserts at a density of 10(6) cells/cm2 (day 0). Bioelectric properties of cultures grown in an air-interface were compared with those covered by medium. On day 1 for air-interface cultures, apical fluid was removed and basolateral fluid was replenished. For cultures covered by medium, varying volumes of apical fluid were used. On days 4 and 5 after plating, confluent GPTE monolayers in either liquid-covered or air-interface cultures exhibited similar monolayer resistance values > or = 1.0 kohm-cm2. However, the equivalent short-circuit current (Ieq) was significantly higher in air-interface cultures than those covered with medium on days 4 and 5. The Ieq in air-interface cultures on day 4 was 12.9 microA/cm2. These confluent GPTE cell monolayers cultured in air-interface could be a useful tool for studies of changes in bioelectric and ion transport properties in response to injury and mediators of inflammation.