Microspheres were used in earlier studies to determine the distribution of blood flow within the intestinal wall, but the selection of microsphere size was not systematic. In one study, a 5-micrometers difference in mean diameter markedly altered the submucosal vs. mucosal distribution of microspheres. Therefore, we examined the effect of size on microsphere distribution within the gut wall. Loops of canine small bowel were perfused at constant pressure, and microspheres were injected in the following order: 9 +/- 1, 15 +/- 1, and 26 +/- 2 micrometers. Gut segments were separated into mucosal, submucosal, and muscularis tissues. The microspheres were recovered from digested tissues and venous blood. The number and diameter of spheres in an aliquot of resuspended spheres were determined by digital image analysis. The ratio of mucosal to submucosal accumulation varied 60-fold for sphere diameters from 8 to 20 micrometers. The muscularis' share did not vary systematically with sphere size. Approximately 20% of spheres less than 11 micrometers reached venous blood, whereas few (less than 1%) of larger sizes did. We conclude: 1) sphere distribution within the gut wall is size dependent, 2) correction for microsphere shunting will be difficult, because the route taken by untrapped spheres is unknown, and 3) fractionating intramural flow among the three layers is not practical with commercially available spheres. However, under control conditions 15-micrometers spheres seem to distribute independently of size between muscularis and a lumped mucosal-submucosal compartment.