Male prairie dogs received in standard diets either 0.08% cholesterol (control, n = 30) or 1.2% cholesterol (lithogenic, n = 31). Animals were killed at days 2-4, 7, 10, 21, and 39 to determine the temporal sequence of changes in mucosal cyclic adenosine 3':5'-monophosphate in the gallbladder and of cholesterol saturation, glycoproteins, cholesterol crystals, and gallstones in bile of prairie dogs fed a cholesterol-rich lithogenic diet. Glycoprotein concentration in bile in the lithogenic group was significantly elevated compared to controls on all days of death. Saturation of bile and formation of cholesterol crystals occurred only in the lithogenic group, detected first after 7 days of feeding. Gallstones were found in the lithogenic group only. Elevation of cyclic adenosine 3':5'-monophosphate in the mucosa of gallbladders was found in the lithogenic group only, beginning at day 10. In summary, increased glycoproteins in bile preceded cholesterol saturation and crystallization which, in turn, preceded increased mucosal cyclic adenosine 3':5'-monophosphate.