We isolated segments of sheep bronchioles (length 406 +/- 46 microns, diameter 227 +/- 53 microns, n = 27). These segments were placed in a 37 degree C bath and cannulated at both ends with glass micropipettes. The proximal end was cannulated and held by three concentrically arranged micropipettes that delivered perfusion solution by gravity. The distal end was cannulated and held by two concentrically arranged micropipettes for fluid collection. When both the lumen and the bath contained oxygenated Krebs-Henseleit buffer (pH 7.40), spontaneous potential difference (PD) was 2.46 +/- 0.39 mV, lumen negative. KCN in the bath diminished PD by 1.48 +/- 0.29 mV (P = 0.00003, n = 25). The isolated bronchiole was depolarized when the luminal solution was Na free (delta PD -1.78 +/- 0.11 mV, P = 0.00007, n = 5) or when the submucosal bath was Cl free (delta PD -2.63 +/- 0.81, P = 0.018, n = 7). In preparations pretreated with 10 microM indomethacin, 1 microM isoproterenol raised PD by 0.75 +/- 0.29 mV (P = 0.03, n = 8), whereas 0.1 mM submucosal bumetanide reduced PD by 0.21 +/- 0.04 mV (P = 0.0005, n = 8). The data show that microperfusion technique is applicable for investigating ion transport by distal bronchioles and that the bronchiolar epithelium probably has both Na and Cl conductive pathways.