OBJECTIVES: Hydrophobicity is the most important determinant of toxicity of bile acids (BAs) and depends on the number, position and orientation of hydroxyl groups. Ursodeoxycholic acid (UDCA) is a hydrophilic dihydroxy BA, which is formed by 7β-epimerization of chenodeoxycholic acid (CDCA) in the gut by intestinal bacteria. Unlike the other secondary BAs, UDCA exerts antiapoptotic effects by preventing oxidative stress. The aim of our study was to analyze the influence of stereochemistry of hydroxyl groups on antiproliferative activity of BAs. METHODS: Human colon adenocarcinoma HT-29 cells were used to assess the cytotoxicity of CDCA and UDCA using colorimetric MTT assay. In order to explain obtained results of MTT assay, 12 molecular descriptors relevant to polarity, solubility and membrane transport were calculated from 3D structures of CDCA and UDCA using VolSurf+ software. RESULTS: Studied BAs displayed distinct degrees of cytotoxicity towards HT-29 cancer cells in a concentration-dependent manner. Concentrations of CDCA and UDCA that inhibited cell growth by 50% (IC) were 19.6 μM and 351.9 μM, respectively. Oxidative stress is considered to be the most plausible mechanism of cytotoxicity of BAs, which is determined mostly by their hydrophobicity. Calculated molecular descriptor that may explain these distinct cytotoxic effects is amphiphilic moment (A), which is defined as a vector pointing from the centre of hydrophobic domain to the centre of hydrophilic domain. The vector length (5.62 of CDCA and 4.87 of UDCA) determines the ability of compound to permeate a membrane. This was additionally substantiated with the values of CACO2, SKIN and LgBB descriptors that indicate higher Caco-2 permeability, skin permeability and blood-brain barrier distribution of CDCA in comparison to UDCA. CONCLUSIONS: More pronounced antiproliferative activity of CDCA in comparison to UDCA was observed. We suggest that computational methods exploring the physicochemical properties of molecules may help in prediction of their cytotoxicity.