The effects of age and dietary fat on bile cholesterol saturation index (CSI) and cholesterol gallstone formation were studied in three age groups of male African green monkeys. Animals were fed semipurified diets containing cholesterol (0.34 g/100 g diet) and 42% of energy as saturated or polyunsaturated fat. Animals were killed at 16 (pre-pubertal), 32 (peri-pubertal) or 60 (young adult) mo of age; the presence of gallstones was determined and gallbladder bile was analyzed for CSI and bile acid composition. Cholesterol gallstones were present in 9% of 60-mo-old animals, compared with a 42% incidence in adult animals fed these same diets for 5 y. Gallbladder bile from the 32-mo-old group contained significantly more cholesterol and had a higher CSI as compared with the other age groups. Dietary fat had no effect on biliary lipid composition. We conclude that the increase in CSI around the time of puberty in male monkeys is not clinically significant in terms of gallstone formation, and that polyunsaturated fat feeding is not associated with adverse effects. Because the incidence of gallstone formation in these young adults was lower than that seen in older adults, a minimal amount of time consuming a lithogenic diet as an adult seems to be required for stone genesis.