The extracellular space (ECS) is the aqueous matrix surrounding cells in solid tissues. The only method to measure ECS volume fraction (alpha) in vivo has been tetramethylammonium iontophoresis, a technically challenging method developed more than 25 years ago. We report a simple, quantitative method to measure alpha by microfiberoptic fluorescence detection of a self-quenched green dye, calcein, and a reference red dye, sulforhodamine 101, after pulsed iontophoretic infusion. The idea is that the maximum increase in calcein fluorescence after iontophoresis is proportional to the aqueous volume into which the dye is deposited. We validated the method theoretically, and experimentally, using cell-embedded gels with specified alpha and ECS viscosity. Measurements in living mice gave alpha of 0.20 +/- 0.01 in brain, 0.13 +/- 0.02 in kidney and 0.074 +/- 0.01 in skeletal muscle. The technical simplicity of the "pulsed-infusion microfiberoptic photodetection" method developed here should allow elucidation of the relatively understudied biological roles of the ECS.