Particle uptake into intestinal tissue has seen increasing attention due to its implications in drug delivery. We attempted to observe a delivery system in vivo and examine uptake in different species. Microspheres were fabricated from polymers including polyanhydrides and delivered to an isolated loop of intestine in several species. The microspheres contained a dye either conjugated to a protein or incorporated freely and were used to qualitatively detect and locate the spheres in the villi of the length of the small intestine. Microspheres were dispersed, sized by a Coulter particle size analyzer, and characterized by confocal and cross-polarized light microscopy, FTIR and SEM. Coulter analysis revealed microspheres to be generally less than 5 microns in diameter. SEM typically showed homogeneous morphology among groups of microspheres. In vivo uptake experiments were performed in rodents, pigs, and ruminants using various microsphere formulations. Microspheres were delivered into the proximal end of the jejunum of anesthetized animals and allowed adequate transit time to be taken up. Animals were euthanized at various time points for explantation of tissue and sampling of blood. Excised samples were embedded inq polyvinyl alcohol, frozen, and cut into sections ranging between 7 and 14 μm in thickness. Our method of incorporating dyes allowed for simultaneous visualization by visible light microscopy and confocal laser scanning microscopy. Two-fluorochrome fluorescence of the microspheres and optical sectioning confirmed the presence of microspheres within intestinal tissue. The amount of uptake depended on the animal model, the duration of the experiment, and the composition of the microsphere. An assay for either the fluorescent dye, the protein attached to it, or the polymer encapsulating it may enable us to determine intracellular concentrations of mierospheres for the quantification of uptake.