Mucin glycoproteins from the gallbladder epithelium are thought to contribute to the matrix or nucleus of gallstones and other biomineralization systems. The involved acidic glycoproteins have been reported in bile and gallstones. In addition, osteopontin (Opn) is a noncollagenous acidic bone matrix glycoprotein that possesses calcium-binding properties. To investigate the role of Opn in pigment gallstone formation, the involvement of Opn in pigment gallstone formation was studied immunohistochemically in the gallbladder wall and in the stones. Staining for Opn was strongly positive in the epithelium of stone-laden gallbladders and in their stones. The stone-laden gallbladders were infiltrated by macrophages, which intensely stained for Opn. Sections of the pigment stones, under low magnification, showed a lamellar pattern of Opn immunolabeling and showed a reticular pattern under high magnification. Our results indicate that Opn, an acidic glycoprotein from the gallbladder epithelium, seems to be involved in lithiasis. Opn from macrophages and/or the epithelium seems to help form the matrix protein.