Although in the last few decades, titanium dioxide nanoparticles (TiO₂NPs) have attracted extensive interest due to their use in wide range of applications, their influences on human health are still quite uncertain and less known. Evidence exists indicating TiO₂NPs ability to enter the brain, thus representing a realistic risk factor for both chronic and accidental exposure with the consequent needs for more detailed investigation on CNS. A rapid and effective in vitro test strategy has been applied to determine the effects of TiO₂NPs anatase isoform, on human glial (D384) and neuronal (SH-SY5Y) cell lines. Toxicity was assessed at different levels: mitochondrial function (by MTT), membrane integrity and cell morphology (by calcein AM/PI staining) after acute exposure (4-24-48 h) at doses from 1.5 to 250 μg/ml as well as growth and cell proliferation (by clonogenic test) after prolonged exposure (7-10 days) at sub-toxic concentrations (from 0.05 to 31 μg/ml). The cytotoxic effects of TiO₂NPs were compared with those caused by TiO₂ bulk counterpart treatment. Acute TiO₂NP exposure produced (i) dose- and time-dependent alterations of the mitochondrial function on D384 and SH-SY5Y cells starting at 31 and 15 μg/ml doses, respectively, after 24h exposure. SH-SY5Y were slightly more sensitive than D384 cells; and (ii) cell membrane damage occurring at 125 μg/ml after 24h exposure in both cerebral cells. Comparatively, the effects of TiO₂ bulk were less pronounced than those induced by nanoparticles in both cerebral cell lines. Prolonged exposure indicated that the proliferative capacity (colony size) was compromised at the extremely low TiO₂NP doses namely 1.5 μg/ml and 0.1 μg/ml for D384 and SH-SY5Y, respectively; cell sensitivity was still higher for SH-SY5Y compared to D384. Colony number decrease (15%) was also evidenced at ≥0.2 μg/ml TiO₂NP dose. Whereas, TiO₂ bulk treatment affected cell morphology only. TiO₂ internalization in SH-SY5Y and D384 cells was appreciated using light microscopy. These findings indicated, that (i) human cerebral SH-SY5Y and D384 cell lines exposed to TiO₂NPs were affected not only after acute but even after prolonged exposure at particularly low doses (≥ 0.1 μg/ml), (ii) these in vitro critical doses were comparable to literature brain Ti levels detected in lab animal intranasally administered with TiO₂NP and associated to neurotoxic effects. In summary, the applied cell-based screening platform seems to provide effective means to initial evaluation of TiO₂NP toxicity on CNS.