Arsenic trioxide (As2O3) is an anticancer drug used in humans to treat some forms of cancer. However its clinical application could also result in secondary effects such as neuro-, hepato- or nephro-toxicity. As2O3 interactions with cells are not fully understood, but effects on calcium signalling could be a major factor of As2O3 interactions with living cells. In this work it was investigated whether As2O3 influences the intracellular calcium ([Ca2+]i) homeostasis in two human derived cells lines: human neuroblastoma SY-5Y and embryonic kidney cells (HEK 293) and whether As2O3 induced [Ca2+]i modifications are related to cytotoxicity. To measure Ca2+ changes during the application of As2O3 calcium sensitive dyes (fluo-4 and rhod-2) were used combined with laser scanning microscopy or fluorescence activated cell sorting. In addition cytotoxicity tests were employed (Trypan blue extrusion and MTT assays). As2O3 (1 µM) increased [Ca2+]i in SY-5Y and HEK 293 cells. Three forms of [Ca2+]i elevations were found: (1) steady-state increases, (2) transient [Ca2+]i–elevations and (3) Ca2+-spikes. [Ca2+]i modifications were independent from extracellular Ca2+ but dependent on internal calcium stores. The effect was not reversible. As2O3 is able to modulate calcium signalling even with a low concentration of 100 pM. Steady state increase of [Ca2+]i and calcium-spikes were observed. Calcium rise depended on time and drug concentration. Inositol triphosphate (IP3) and ryanodine (Ry) receptors are involved in regulation of calcium signals induced by As2O3. In addition, cyclosporine A sensitive calcium pools are similarly modulated in neuroblastoma and HEK cells, while the caffeine and ryanodine sensitive calcium pools are differentially regulated in the two cell line. In cytotoxicity tests As2O3 (1µM) significantly reduced cell viability in both cell types. Staining with Hoechst 33342 showed occurrence of apoptosis and DNA damage with 1µM As2O3. Lower concentrations have a specific apoptotic effect for neuroblastoma cells but not for HEK cells. The data suggest that [Ca2+]i is an important messenger in As2O3 induced cell death and that low concentrations of As2O3 are able to interfere with physiological processes in diverse cell models.