To systematically study airway epithelial barrier properties and active ion transport processes during challenge to environmental toxicants, in vitro models that closely resemble airway epithelium in vivo are required. Guinea pig tracheobronchial epithelial (GPTE) cells cultured in an air interface form a tight, confluent monolayer, which may be a more suitable model for the studies of the airway epithelial barrier in vivo. In the present study, bioelectric properties of such GPTE cell monolayers were characterized with the use of pharmacological agents. Treatment of the basolateral side with ouabain completely abolished the short-circuit current (SCC), while apical addition had little effect. Apical addition of amiloride abolished 75% of the SCC, while basal addition had little effect. Treatment of the basolateral side with furosemide or bumetanide reduced the SCC by 20%, while apical addition had no effect. Apical or basolateral terbutaline increased SCC threefold with identical time courses; results with furosemide suggest a preferential stimulation of Cl- secretion. In contrast to GPTE monolayers, canine tracheal epithelial monolayers, under short-circuit conditions, primarily secrete Cl-, possibly due to apparent species differences. The predominance of Na+ reabsorption suggests that GPTE monolayers cultured in an air interface may share similar transport properties to human airway epithelium.