In this study we aimed to (1) screen phenothiazines for cytotoxic activity in glioma, neuroblastoma, and primary mouse brain tissue; and (2) determine the mechanism of the cytotoxic effect (apoptosis, necrosis) and the roles of calmodulin inhibition and σ receptor modulation. Rat glioma (C6) and human neuroblastoma (SHSY-5Y) cell lines were treated with different phenothiazines. All agents induced a dose-dependent decrease in viability and proliferation, with the highest activity elicited by thioridazine. Sensitivity to thioridazine of glioma and neuroblastoma cells was significantly higher (p<0.05) than that of primary mouse brain culture (IC₅₀ 11.2 and 15.1 µM vs 41.3 µM, respectively). The N-mustard fluphenazine induced significantly lower cytotoxicity in glioma cells, compared to fluphenazine. The σ receptor selective ligand (+)-SK&F10047 increased viability slightly while combined with fluphenazine; SK&F10047 did not alter fluphenazine activity. Flow cytometry of propidium iodide (PI)-stained glioma cells treated with thioridazine, fluphenazine, or perphenazine (6–50 µM) resulted in a concentration-dependent increase of fragmented DNA up to 94% vs 3% in controls by all agents. Thioridazine (12.5 µM)-treated glioma cells costained with PI and Hoechst 33342 revealed a red fluorescence of fragmented nuclei in treated cells and a blue fluorescence of intact control nuclei. After 4-h exposure to thioridazine (25 and 50 µM), a 25- to 30-fold increase in caspase-3 activity in neuroblastoma cells was noted. Overall, the marked apoptotic effect of phenothiazines in brain-derived cancer cells, and the low sensitivity of primary brain tissue suggest the potential use of selected agents as therapeutic modalities in brain cancer.