The fiber matrix theory of capillary permeability was evaluated by measuring permeability coefficients to colored solutes with Stokes radii between 0.5 and 1.76 nm. In vivo calibration of a microscope photometer established the range of linearity for optical density measurements from a rectangular window that included the test capillary and surrounding tissue. In individually perfused capillaries of frog mesentery the following permeability coefficients, expressed as mean +/- SE X 10(5) cm/s were obtained: azure C (mol wt 277), 11.0 +/- 0.2; patent blue violet (566), 3.95 +/- 0.67; Evans blue (960), 1.15 +/- 0.23; microperoxidase (1,900), 1.13 +/- 0.31; and FITC-dextran 3 (3,400), 0.48 +/- 0.18. There is significant restriction to diffusion of all solutes larger than azure C. Fibers 0.5 nm in radius occupying 8.5% of the volume of intercellular junctions (area 2.2 X 10(-3) cm2/cm2, depth 0.6 X 10(-4) cm) account for the 22-fold range of measured permeabilities. The fiber density estimated is larger than that required to describe water flow through the junction. Negative charges on solutes larger than 0.5 nm radius may contribute to exclusion and restriction to diffusion in intercellular junctions.