A non-obstructing optical method was developed to measure proximal tubular fluid reabsorption in rat nephron at 0.25 Hz . The effects of uncaging luminal NO on proximal tubular reabsorption were investigated with this method. Proximal fluid reabsorption rate was calculated as the difference of tubular flow measured simultaneously at two locations (0.8-1.8 mm apart) along a convoluted proximal tubule. Tubular flow was estimated based on the propagating velocity of fluorescent dextran pulses in the lumen. Changes in local tubular flow induced by intratubular perfusion were detected simultaneously along the proximal tubule, indicating that local tubular flow can be monitored in multiple sites along a tubule. The estimated tubular reabsorption rate was 5.52 ± 0.38 nl/min/mm (n=20). Flash photolysis of luminal caged NO (potassium nitrosylpentachlororuthenate) was induced by a 30 Hz ultraviolet nitrogen-pulsed laser. Release of NO from caged NO into the proximal tubule was confirmed by monitoring intracellular [NO] using a cell-permeant NO-sensitive fluorescence dye (DAF-FM). Emission of DAF-FM was proportional to the number of laser pulses used for uncaging. Photolysis of luminal caged NO induced a dose dependent inhibition of proximal tubular reabsorption without activating tubuloglomerular feedback, while uncaging of intracellular cGMP in proximal tubule decreased tubular flow. Coupling of this novel method to measure reabsorption with photolysis of caged signaling molecules provides a new paradigm to study tubular reabsorption with ambient tubular flow.