The mechanism of proximal tubule potassium reabsorption was studied by stopped-flow microperfusion and determination of potassium activities by ion-sensitive resin microelectrodes. The proximal tubule was unable to establish transepithelial potassium gradients. Perfusion with 20 mM K+ turned the lumen 3 mV more negative, an effect abolished by Ba2+. The half-time for K+ activities to reach their stationary level after perfusion with 1 mM K+ was significantly increased by Ba2+ from 4.25 +/- 0.14 s to 11.0 +/- 1.71 s, and after perfusion with 20 mM K+, from 5.43 +/- 0.20 to 12.53 +/- 0.90 s. These data indicate that a significant fraction of potassium is transferred across proximal tubule epithelium by a transcellular, K(+)-channel-dependent route.