Deoxycholic acid (DCA) and other hydrophobic bile acids induce apoptosis at submicellar concentrations, while bile acids such as ursodeoxycholic acid (UDCA) and its taurine conjugate (TUDCA) display cytoprotective properties. The mechanisms that trigger these opposite signalling effects are still unclear. Recent studies have confirmed that cytotoxic bile acids decrease membrane order in membrane model systems and in purified plasma membrane vesicles, suggesting that cytotoxicity could be initiated through modulation of plasma membrane structure. Using confocal microscopy and two-photon fluorescence microscopy of Laurdan in hepatocytes and established cell lines, we have shown that upon uptake, bile acid analogues accumulate in intracellular membranes and display remarkable low plasmalemmal levels. Incubation of hepatocytes with both classes of bile acids resulted in a dramatic decrease in intracellular membrane order, as a result of bile acid accumulation during uptake. Bile acids also accumulated in mitochondria, but only DCA induced significant changes in the membrane order of isolated mitochondria. Importantly, the DCA induced changes in membrane fluidity preceded an increase in mitochondrial permeability, which is not detected for both UDCA and TUDCA. Our results are consistent with the presence of cellular compensatory mechanisms, which work against the moderate loading of bile acids in the plasma membrane, but that are unable to balance the increase in membrane fluidity induced by cytotoxic bile acids in mitochondrial membranes. Our findings suggest that apoptosis induced by cytotoxic bile acids is the result of changes in mitochondrial membrane structure after incorporation of bile acids.Supported from PTDC/QUI-BIQ/119494/2010 and RECI/CTM-POL/0342/2012.T.S acknowledges FCT grant SFRH/BD/92398/2013