The paracellular pathway permeability is known to increase in perfused amphibian kidneys if the luminal fluid is made hyperosmotic with mannitol or urea. To investigate whether luminal hypertonicity increases paracellular pathway permeability in the mammalian nephron, early rat distal tubules were micropunctured and perfused through one micropipette with either isosmotic saline (IS), hyperosmotic urea (HU) or hyperosmotic mannitol (HM) solutions. A second micropipette was placed down-stream in the same tubule and test solutions of 30 nl of a mixture of 14C-inulin and 3H-mannitol or of 3H-inulin and 14C-urea were injected. Similar intratubular injections of tracers were performed in a second group of rats undergoing diuresis induced either by infusing intravenously saline alone (VS) or receiving saline plus 0.4 M urea (VU). In the latter group (VU) luminal urea concentration was increased without the tubular lumen being made hyperosmotic to its peritubular fluid. Urinary unulin recovery was essentially complete and unaffected by experimental procedures. Difference between mannitol recoveries in isosmotic saline and hyperosmotic urea perfusions IS-HU was 2.6 +/- 0.8% (P less than 0.001). Difference in urea recoveries IS-HM was 4.1 +/- 5.1% (P greater than 0.40), IS-HU was 13.9 +/- 5.3% (P equal to 0.015) and, VS-VU equal to 17.0 +/- 3.4 (P less than 0.001). Therefore, elevated luminal urea concentration increased tracer mannitol and also tracer urea permeability, both in the presence and absence of tubular hyperosmolarity. Electron microscopic observations showed changes in geometry of tubular junctional complexes compatible with the observed increase in permeability.