An intravital fluorescence microscopic method and digital image analysis were established for measuring the changes of fluorescein sodium (FINa, MW 376) and FITC-Dextrans (FD-4, MW4000, FD-150, MW150000) permeability in mesenteric microvessels. The transport of various molecular fluorescent tracer within the blood vessels and through the vessel walls into the perivascular tissue of the rat mesentery was studied using a highly sensitive CCD camera, the fluorescence intensities were transformed into video signals and recorded for subsequent analysis. The results showed that FINa could pass through the wall of microvessels very quickly. The diffusion constant was 3.362 x 10(-8) cm2/s with FINa, and FD-4 was 1.718 x 10(-8) cm2/s, FD-150 was 0.0486 x 10(-8) cm2/s. The diffusion constant depended very strongly on the molecular weight and showed no difference between capillary and vessels of the venous part of the microcirculation, but in vessels of the arterial part of microcirculation no diffusion was found. That was to say that diffusion occurred in capillary and microvenous, but not in microarterial. It is expected that this method would be very useful for the quantitive analysis in various molecular weights and in monolayer endothelial cell permeability.