We studied the detailed localization of hyaluronic acid in the seminal vesicles of the miniature pig, using hyaluronic acid-binding protein as a specific histochemical probe at the ultrastructural level. According to the results, the basolateral surface of the plasma membrane of the glandular epithelial cells, was found to contain hyaluronan. However, abundantly present was hyaluronan in the subepithelial connective tissue, in particular, in the extracellular matrix surrounding the fibroblasts, smooth muscle cells, small blood vessels and capillaries. The substance was also observed in the surface coat of the plasma membrane of the fibroblasts, but not in that of the smooth muscle cells. The findings suggest that hyaluronan in the seminal vesicles of the miniature pig is synthesized onto the surface coat of the plasma membrane of the fibroblasts, is contributed to the extracellular matrix, and consequently concentrates in the subepithelial connective tissue. The substance may particularly be involved in a variety of cellular functions to maintain morphological organization as well as to regulate physiological homeostasis in the reproductive organ of this species, rather than participate in sperm functions.