Previous studies from our laboratory indicate that early distal segments of the rat kidney have a positive transepithelial potential difference (PD). The present study investigates the origin of the positive PD. PDs were measured in early distal segments using a technique which allowed simultaneous microperfusion and PD measurement through a single pipette (3 to 6 micron O.D.). Microperfusion with artificial plasma ultrafiltrate resulted in a significantly negative mean PD of -4.9 +/- 0.7 mV (N = 17), in contrast to a positive free-flow PD of +5.7 +/- 1.1 mV (N = 174) (P less than 0.001). Addition of amiloride 10(-4) M to plasma ultrafiltrate changed the PD to +1.7 +/- 0.2 mV (N = 25, P less than 0.001). In contrast, furosemide 10(-4) M had no effect on the perfusion PD. Removal of sodium from the luminal perfusate abolished any effect of amiloride on the perfusion PD. Perfusion with artificial early distal fluid yielded a positive PD of +4.2 +/- 0.2 mV (N = 19). Amiloride increased this PD to +8.3 +/- 0.7 mV (N = 21, P less than 0.001). Subsequent experiments in which the sodium and potassium concentrations of the perfusates were varied indicated that concentration gradients for these ions across the early distal tubule could generate substantial diffusion PDs and that potassium was much more permeant than sodium.(ABSTRACT TRUNCATED AT 250 WORDS)