This study identified mucus granules, determined mode of release and quantified their volume in the gallbladder epithelium of Richardson's ground squirrels (Spermophilus richardsonii) fed a lithogenic diet of 2% cholesterol to experimentally induce gallstone formation. Tissue was examined using light microscopy histochemistry, scanning and transmission electron microscopy, as well as autoradiography and morphometry at the electron microscopic level. Autoradiography demonstrated incorporation of a glycoprotein precursor, [3H]galactose, within the membrane-bound granules localized in the supranuclear region of the epithelial cells. Exocytosis of granule contents was by merocrine secretion. Morphometry indicated a significant increase in the amount of intracellular mucin granules as early as 18 hr on the lithogenic diet, a feature that continued throughout the experimental period of 20 weeks. Mucus synthesis/secretion rates returned to control values within 3 weeks after removal from the diet. Scanning electron microscopy observations revealed a thick sludge-like layer overlying the epithelium at a time in the chronology of the cholelithiasic model that correlated well with the initial phases of stone formation. Histochemistry showed this layer to be a mixture of acidic mucins. Neutral mucins were not observed. The hypersecretion of mucus and formation of this sludge-like layer appear to be critical nucleating factors in the formation of cholesterol gallstones.