Bile acids (BAs) are core gastrointestinal metabolites with dual functions in lipid absorption and cell signaling. BAs circulate between the liver and distal small intestine (i.e., ileum), yet the dynamics through which complex BA pools are absorbed in the ileum and interact with host intestinal cells in vivo remain poorly understood. Because ileal absorption is rate-limiting in determining which BAs in the intestinal lumen gain access to host intestinal cells and receptors, and at what concentrations, we hypothesized that defining the rates and routes of ileal BA absorption in vivo would yield novel insights into the physiological forms and functions of mouse enterohepatic BA pools.