Transmembrane potential difference (pd) was studied in isolated perfused segments of rabbit proximal convoluted tubules. At perfusion flow rates above 10 nl/min the pd was -5.80 ±0.3 mv (lumen negative) when perfusing with isosmolal ultrafiltrate of same rabbit serum as the bath. That this pd is generated by transport activity of the tubule is supported by three separate observations: (a) pd reversibly decreased with cooling from 37°C to 25°C; (b) pd decreased when 10^-5 M ouabain was added to the bath and reversed to control levels when ouabain was removed; and (c) heating to 47°C irreversibly decreased pd to zero. The magnitude of the pd was related to perfusion flow rate at slower rates than 10 nl/min. A decrease in flow rate was associated with a decrease in pd. The tubular geometry and transmembrane hydrostatic pressure were ruled out as the mediating factors governing the magnitude of observed pd.