Quantitative trait locus (QTL) mapping was used to locate genes that determine the difference in cholesterol gallstone disease between the gallstone-susceptible strain C57L/J and the gallstone-resistant strain AKR/J. Gallstone weight was determined in 231 male (AKR x C57L) F(1) x AKR backcross mice fed a lithogenic diet containing 1% cholesterol, 0.5% cholic acid, and 15% butterfat for 8 wk. Mice having no stones and mice having the largest stones were genotyped at approximately 20-cM intervals to find the loci determining cholesterol gallstone formation. The major locus, Lith1, mapped near D2Mit56 and was confirmed by constructing a congenic strain, AK. L-Lith1(s). Another locus, Lith2, mapped near D19Mit58 and was also confirmed by constructing a congenic strain AK.L-Lith2(s). Other suggestive, but not statistically significant, loci mapped to chromosomes 6, 7, 8, 10, and X. The identification of these Lith genes will elucidate the pathophysiology of cholesterol gallstone formation.