To assess whether proximal luminal fluid becomes hypotonic with respect to plasma, free-flow micropuncture measurements were made sequentially from the end-proximal tubule to Bowman's space in 10 tubules of hydropenic Munich-Wistar rats. Osmolality in Bowman's space was 2.8 +/- 0.3 mosmol less than in plasma. Tubular fluid osmolality fell along the tubule and by the end-proximal tubule was 7.5 +/- 0.7 mosmol/kg less than in plasma or 4.7 mosmol/kg less than in Bowman's space. Since luminal fluid became hypotonic, the reabsorbate was hypertonic. The transepithelial osmotic water permeability (Pf) was calculated using simultaneously measured water reabsorption rates. The osmotic gradient responsible for water reabsorption was assumed to be either lumen-to-reabsorbate or lumen-to-peritubular plasma, with a reflection coefficient for sodium chloride of 0.7-1.0. The Pf was then estimated to be between 0.2 and 2.0 cm/s in the first millimeter of tubule and to have fallen to 0.1-0.2 cm/s by the end of the tubule. In conclusion, luminal hypotonicity develops in the rat proximal convoluted tubule and must be considered as part of the osmotic driving force for water reabsorption.