Whole-cell patch clamp techniques were used to investigate the amiloride-sensitive sodium conductance (G_Na) in the everted initial collecting tubule of Ambystoma. Accessibility to both the apical and basolateral membrane made this preparation ideal for studying the regulation of sodium transport by insulin. G_Na accounted for 20% of total cell conductance (G_T) under control conditions. A resting membrane potential of -75 ± 2 mV (7) together with the fact that G_T is stable with time suggested the cells studied were viable. Measurements of capacitance and use of a known uncoupling agent, heptanol, suggested cells were not electrically coupled. Thus the values of G_T and G_Na represented individual principal cells. Exposure of the basolateral membrane to insulin (1mU/ml) for 10-60 minutes significantly (p<0.05) increased the normalized G_Na [1.2 ± 0.3 nS (6) vs. 2.0 ± 0.4 nS (6)]. Cell-attached patch clamp techniques were utilized to further elucidate the mechanism by which insulin increased amiloride-sensitive epithelial sodium channel (ENaC) activity. In the presence of insulin there was no apparent change in either the number of active levels/patch or the conductance of ENaC. The open probability increased significantly (p<0.01)from 0.21 ± 0.04 (6) to 0.46 ± 0.07 (6). Thus, application of insulin enhanced sodium reabsorption by increasing the fraction of time the channel spent in the open state.