Humans are exposed to distinct structural classes of insecticides with different neurotoxic modes of action. Since calcium homeostasis is essential for proper neuronal function and development, we investigated the effects of insecticides from different classes (pyrethroid: (α-)cypermethrin; organophosphate: chlorpyrifos; organochlorine: endosulfan; neonicotinoid: imidacloprid) and mixtures thereof on the intracellular calcium concentration ([Ca(2+)]i). Effects of acute (20 min) exposure to (mixtures of) insecticides on basal and depolarization-evoked [Ca(2+)]i were studied in vitro with Fura-2-loaded PC12 cells and high resolution single-cell fluorescence microscopy. The data demonstrate that cypermethrin, α-cypermethrin, endosulfan, and chlorpyrifos concentration-dependently decreased depolarization-evoked [Ca(2+)]i, with 50% (IC50) at 78 nM, 239 nM, 250 nM and 899 nM, respectively. Additionally, acute exposure to chlorpyrifos or endosulfan (10 μM) induced a modest increase in basal [Ca(2+)]i, amounting to 68±8 nM and 53±8 nM, respectively. Imidacloprid did not disturb basal or depolarization-evoked [Ca(2+)]i at 10 μM. Following exposure to binary mixtures, effects on depolarization-evoked [Ca(2+)]i were within the expected effect additivity range, while the effect of the tertiary mixture was less than this expected additivity effect range. These results demonstrate that different types of insecticides inhibit depolarization-evoked [Ca(2+)]i in PC12 cells by inhibiting VGCCs in vitro at concentrations comparable to human occupational exposure levels. Moreover, the effective concentrations in this study are below those for earlier described modes of action. Since inhibition of VGCCs appears to be a common and potentially additive mode of action of several classes of insecticides, this target should be considered in neurotoxicity risk assessment studies.