Whole cell patch-clamp techniques were used to investigate amiloride-sensitive sodium conductance (G_Na) in the everted initial collecting tubule of Ambystoma. Accessibility to both the apical and basolateral membranes 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 (n = 7) together with the fact that G_T is stable with time suggested that the cells studied were viable. Measurements of capacitance and use of a known uncoupling agent, heptanol, suggested that 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 (1 mU/ml) for 10-60 min significantly (P < 0.05) increased the normalized G_Na [1.2 ± 0.3 nS (n = 6) vs. 2.0 ± 0.4 nS (n = 6)]. Cell-attached patch-clamp techniques were used to further elucidate the mechanism by which insulin increases 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 (n = 6) to 0.46 ± 0.07 (n = 6). Thus application of insulin enhanced sodium reabsorption by increasing the fraction of time the channel spent in the open state.