Dinitrophenolic compounds are powerful toxicants with a long history of use in agriculture and industry. While (high) human exposure levels are not uncommon, in particular for agricultural workers during the spraying season, the neurotoxic mechanism(s) that underlie the human health effects are largely unknown. We therefore investigated the in vitro effects of two dinitrophenolic herbicides (DNOC and dinoseb) on a battery of neurotoxicity endpoints in (dopaminergic) rat PC12 cells. Cell viability, mitochondrial activity, oxidative stress and caspase activation were assessed using fluorescence-based bioassays (CFDA, alamar Blue, H2DCFDA and Ac-DEVD-AMC, respectively), whereas changes in intracellular [Ca(2+)]i were assessed using single-cell fluorescence microscopy with Fura-2AM. The combined results demonstrate that exposure to both DNOC and dinoseb is linked to calcium release from the endoplasmic reticulum and activation of caspase-mediated apoptotic pathways. In subsequent experiments, immunofluorescent labelling with specific antibodies was used to determine changes in intracellular α-synuclein levels, demonstrating that both DNOC and dinoseb increase levels of intracellular α-synuclein. The combined results indicate that in vitro exposure to DNOC and dinoseb activates pathways that are not only involved in acute neurotoxicity but also in long-term effects as seen in neurodegeneration.