Our early studies showed that tissue diffusion coefficient (Dt) of small solute sodium fluorescein (0.45 nm radius) in frog mesentery is 30% of its free diffusion coefficient (Dfree) in aqueous solution. We test here the hypothesis that because of its size-limiting structure the interstitium would provide larger resistance to larger solute α-lactalbumin (2.0 nm radius). We extended our previous method by using laser-scanning confocal microscopy to measure both solute capillary permeability (P) and solute tissue diffusion coefficients (Dt) from the rate of tissue solute accumulation and the radial concentration gradients around individually perfused microvessels in frog mesentery. pα-lactalbumin was 1.7 ± 0.7 (SD) × 10−6 cm/s (n = 6). Dt/Dfree was 27% ± 5% (SD) (n = 6). This value is comparable to that for small solute sodium fluorescein. Our results indicate that frog mesenteric interstitium is less selective to larger solute α-lactalbumin than the microvessel wall.