Bile acids (BAs) are natural ligands of nuclear receptors, in particular FXR. Whether, in addition to protein-mediated cytosolic-nuclear BA translocation, other mechanisms are involved in the access of BAs to nuclear FXR was investigated. When rat hepatocytes were incubated with radiolabelled TCA, TDCA, TCDCA, and TUDCA, their nuclear accumulation was proportional to their intracellular levels. Using flow cytometry analysis the accumulation by nuclei isolated from rat liver cells was found to differ for several fluorescent compounds of similar molecular weight and different charge, including fluorescein-tagged BAs (CGamF, UDCGamF or CDCGamF). Varying nuclear volume by incubation with different sucrose concentrations, a similar relationship between nuclear volume and content of FITC and 4 kDa-FITC-dextran was found. In contrast, this relationship was markedly lower for CGamF. Confocal microscopy studies revealed that fluorescein-tagged BAs, but also FITC and 10 kDa-FITC-dextran were found in the nuclear envelope and concentrated in regions where DNA was less densely packed. In contrast to the cytosolic subcellular localization of PPAR, FXR and nucleolin (a marker of transcriptional active chromatin), were also localized by immunoreactivity in these intranuclear regions. In conclusion, although intranuclear levels of small organic molecules, including conjugated BAs, depend on their concentrations in the extranuclear space, the existence of certain molecular selectivity (not strictly dependent on molecular weight or charge) suggests that, in addition to simple diffusional exchange, other mechanisms may be also involved in determining their overall nuclear content in regions where these compounds coincide and may interact with nuclear receptors, such as FXR.