In this work, we present a complete physicochemical characterization of multi-wall carbon nanotubes (mwCNTs) in order to assess their potential toxicological effects in in vitro cell models using Colony Forming Efficiency (CFE) assay. We verified that Dimethyl Sulfoxide (DMSO) was a more suitable solvent to disperse mwCNTs compared to culture medium guaranteeing reproducibility in the preparation of testing dilutions. The CFE assay was carried out on five mammalian cell lines representing the potentially exposed and/or target organs for nanomaterials (lung, liver, kidney, intestine, skin), as well as on mouse fibroblasts cell line, which usually is considered a sensitive model to verify in vitro cytotoxicity of test compounds. A statistically significant toxic effect was found only in human alveolar basal epithelial cells and immortalized mouse fibroblasts, for which the interaction between mwCNTs and cells was additionally studied by Atomic Force and Scanning Electron Microscopy. In this study, we considered and suggested the CFE assay as a promising test for screening studies of cytotoxicity. In addition, combining in vitro tests with physicochemical analysis, this work underlines basic points to be considered when research on nanomaterials has to be carried out, to set up, in our opinion, well-defined and suitable experimental planning and procedures. ; JRC.I.4-Nanobiosciences