Cholylamidofluorescein (CamF) has been selected as a fluorescent bile acid scaffold to perform a full characterization of its photophysical properties. In aqueous medium, under nitrogen, the absorption spectrum of CamF was expectedly dependent on pH. Under air, the presence of CO(2) resulted in a partial protonation of the photoactive form, reducing the absorbance of CamF. The fluorescence spectrum of CamF in ethanol (lambda(exc) = 481 nm) showed a broad band with maximum at 518 nm; the fluorescence quantum yield was 0.67, and the fluorescence lifetime was 4.8 ns. Laser flash photolysis of CamF showed the triplet state transient with a broad maximum at ca. 540 nm and a lifetime of 19 mus. Flow cytometric kinetic assay of CamF uptake in real time was performed in suspensions of rat hepatocytes, showing that living hepatocytes accumulated slowly but constantly CamF along the 5-minute experimental period. Besides, intracellular fluorescence of live cells was found to be clearly dependent on the extracellular concentration of CamF. Thus, flow cytometry has allowed us to demonstrate that CamF is specifically taken up by living rat hepatocytes in a concentration-dependent fashion, suggesting the suitability of this molecule for further studies on bile-acid transport in liver cells.