To clarify the question whether solute and water fluxes between pelvic urine and the renal papilla contribute to the medullary accumulation of osmotically active substances and thus to final urine concentration, we measured the osmolarity of urine samples from the papillary tip of rat kidneys during superfusion of the exposed papillae with solutions of widely varying osmotic concentrations. When the osmolarity of the superfusion fluid consisted half of urea and half of sodium chloride, urine osmolarity was observed to change parallel to the bath solution over a certain concentration range (800–2000 mosm/l). The changes of urine concentration occurred within 90 min after the start of the papillary superfusion. Similar results were obtained when the sodium chloride concentration was kept constant at 300 mosm/l and the urea concentration varied to yield bath concentrations up to 3000 mosm/l. A rise of urine concentration by papillary superfusion above 2000 mosm/l was achieved when exogenous arginine-vasopressin was infused intravenously suggesting that the failure of urine concentration to equilibrate with the bath concentration was due to a limited water permeability of the collecting ducts. These results suggest that solute and water fluxes between pelvic urine and the renal papilla are a necessary prerequisite to achieve maximal osmotic urine concentrations. In addition, such fluxes may explain the variability of medullary tissue concentrations under various diuretic states.