Cytotoxicity screening is a common technique in drug compound screening for the identification of adverse cellular effects. Nanoparticles may cause interference in these assays. For the interpretation of cytotoxicity data it is important to study also the influence of other factors like pre-treatment of the nanoparticles, the choice of the cell culture medium and type of cell used for testing. Carboxyl polystyrene particles (CPS, 20-1000 nm) were physicochemically characterized and cytotoxicity assessed with seven screening assays in 20 cell lines, which differed in species, growth pattern, cell size, doubling time, embryonic origin and capacity for phagocytosis. Small CPS acted more cytotoxic in all cell lines, larger CPS only in phagocytic cells. Small differences in cytotoxicity were noted between the screening assays. Growth pattern and cell size determined cytotoxicity more than proliferation rate and embryonic origin of cells. Non-adherent cells, cells of mesenchymal origin and with high proliferation rate may be more susceptible to damage by nanoparticles.