In inbred mice, susceptibility to cholesterol gallstone disease is conferred by Lith genes, which in part promote hypersecretion of cholesterol into bile in response to a high-fat/cholesterol/cholic acid (lithogenic) diet. Because cytosolic sterol carrier protein 2 (SCP2) is believed to participate in cellular cholesterol trafficking and is elevated in the liver cytosol of cholesterol gallstone patients, we defined the hepatic expression of SCP2 during cholesterol gallstone formation in gallstone-susceptible C57L and gallstone-resistant AKR mice fed the lithogenic diet. Steady-state cytosolic SCP2 levels in C57L, but not AKR mice increased as a function of time and were correlated positively with biliary cholesterol hypersecretion, cholesterol saturation indices of gall-bladder biles and the appearance of liquid and solid cholesterol crystals leading to gallstone formation. Steady-state mRNA levels increased co-ordinately, consistent with regulation of SCP2 expression at the transcriptional level. Our results suggest that overexpression of SCP2 contributes to biliary cholesterol hypersecretion and the pathogenesis of gallstones in genetically susceptible mice. Because of the different chromosomal localizations of the Lith and Scp2 genes, we postulate that Lith genes control SCP2 expression indirectly.