Studies were designed to examine fluid-phase pinocytosis in proximal tubular cells. Canine proximal tubules were obtained from the band IV of Percoll gradient centrifugation of the dispersed renal cortex, and were seeded on collagen-coated polycarbonate membranes. Integrity of monolayers was confirmed by electrophysiologic measurements, and by scanning electron microscopy. At confluence cell monolayers were studied in Ussing chambers. The rate of transfer of a marker of fluid-phase pinocytosis, Lucifer Yellow CH, from the luminal to the basolateral bath was three times higher than that occurring in the opposite direction. Fluorescence microscopy demonstrated that Lucifer Yellow was trapped exclusively in the vesicular compartment. Electron microscopy of the monolayers incubated with cationized ferritin added to the luminal or to the basolateral both revealed that endocytic vesicles were formed only at the luminal surface. Luminal-to-basolateral transfer of Lucifer Yellow was almost completely blocked at 0 degrees C, and was significantly diminished by K+ depletion. Transcytosis of Lucifer Yellow was stimulated twofold by 1-oleoyl-2-acetyl-glycerol. Transfer of quin-2 acetoxymethylester across the monolayer was used as a marker of the paracellular pathway, demonstrating the lack of directional selectivity of this transport route. In summary, vectorial fluid-phase pinocytosis in proximal tubular cells represents an additional mechanism contributing to fluid transport in this segment of the nephron.