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 7alpha-hydroxylase and delta(4)-3-ketosteroid 5beta-reductase) in the hepatocyte-like cells. We could demonstrate cholesterol 7alpha-hydroxylase activity and its circadian rhythm in the hepatocyte-like cells. Northern blot analysis demonstrated the expression of messenger RNA for the 7alpha-hydroxylase and delta(4)-3-ketosteroid 5beta-reductase in the pancreatic hepatocyte-like cells. To measure the amount of the messenger RNA, we used the competitive polymerase chain reaction method for the 7alpha-hydroxylase. This quantitation revealed the existence of a circadian rhythm of cholesterol 7alpha-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.