Studies were performed to characterize hepatic and intestinal transport, as well as biotransformation during transport, of a spectrum of fluorescent bile acids containing a fluorophore attached to the side chain. The following two classes of compounds were studied: 1) aminofluorescein (amF) coupled directly to the carboxylic group of a bile acid which was cholic, ursodeoxycholic, or cholylglycine; and 2) nitrobenzoxadiazolyl (NBD) coupled to the epsilon-amino group of a lysine conjugated bile acid, which was cholic or ursodeoxycholic. Fluorescein, a cholephilic organic anion, was studied as a control. Fluorescent bile acids were synthesized and their structures confirmed by nuclear magnetic resonance and mass spectrometry. Using the biliary fistula rat, hepatic transport, biotransformation, and choleretic activity were defined; intestinal absorption was assessed by jejunal or ileal perfusion studies. All fluorescent bile acids had hepatic transport maxima about one-sixth that reported for cholyltaurine, but derivatives of cholylglycine were transported best. Bile acids underwent little (<5%) biotransformation during hepatocyte transport. Only the amF conjugate of cholylglycine had normal choleretic activity; other compounds were hypocholeretic or cholestatic. In contrast, fluorescein was well transported, was partly glucuronidated, and had normal choleretic activity. NBD-tagged, but not amF-tagged, bile acids were actively transported by the intestine (ileum > jejunum), and no fluorescent bile acid had passive intestinal permeability; NBD-tagged bile acids were biotransformed during intestinal transport (jejunum > ileum). We conclude that the structure of the fluorophore as well as that of the bile acid influences transport by the hepatocyte and enterocyte. These fluorescent bile acids differ from fluorescein in being impermeable to cell membranes and undergoing little biotransformation during hepatocyte transport. Of these fluorescent bile acids, cholylglycylamF has hepatocyte transport and choleretic properties most closely resembling those of a natural bile acid.