Hepatocyte-like cells have been observed in the pancreas of the rat. We examined the bile acid biosynthetic function of these cells to determine whether they were real hepatocytes. This study investigated the existence of two liver-specific enzymes involved in bile acid biosynthesis (cholesterol 7a-hydroxylase and D4-3-ketosteroid 5b-reductase) in the hepatocyte-like cells. We could demonstrate cholesterol 7a-hydroxylase activity and its circadian rhythm in the hepatocyte-like cells. Northern blot analysis demonstrated the expression of messenger RNA for the 7a-hydroxylase and D4-3-ketosteroid 5b-reductase in the pancreatic hepatocytelike cells. To measure the amount of the messenger RNA, we used the competitive polymerase chain reaction method for the 7a-hydroxylase. This quantitation revealed the existence of a circadian rhythm of cholesterol 7a-hydroxylase messenger RNA in the hepatocyte-like cells. These results indicated that bile acid biosynthesis was performed in the pancreatic hepatocyte-like cells as noted as in the liver parenchymal cells. —Ando, Y., H. Ide, S. Kosai, R. Kamimura, Y. Maeda, S. Higashi, and T. Setoguchi. Expression of cholesterol 7-hydroxylase and D4-3-ketosteroid 5b-reductase genes in rat pancreatic hepatocyte-like cells.