alpha-Synuclein is a major protein component deposited in Lewy bodies and Lewy neurites that is extensively phosphorylated at Ser(129), although its role in neuronal degeneration is still elusive. In this study, several apoptotic pathways were examined in alpha-synuclein-overexpressing SH-SY5Y cells. Following the treatment with rotenone, a mitochondrial complex I inhibitor, wild type alpha-synuclein-overexpressing cells demonstrated intracellular aggregations, which shared a number of features with Lewy bodies, although cells overexpressing the S129A mutant, in which phosphorylation at Ser(129) was blocked, showed few aggregations. In wild typealpha-synuclein cells treated with rotenone, the proportion of phosphorylated alpha-synuclein was about 1.6 times higher than that of untreated cells. Moreover, induction of unfolded protein response (UPR) markers was evident several hours before the induction of mitochondrial disruption and caspase-3 activation. Eukaryotic initiation factor 2alpha, a member of the PERK pathway family, was remarkably activated at early phases. On the other hand, the S129A mutant failed to activate UPR. Casein kinase 2 inhibitor, which decreased alpha-synuclein phosphorylation, also reduced UPR activation. The alpha-synuclein aggregations were colocalized with a marker for the endoplasmic reticulum-Golgi intermediate compartment. Taken together, it seems plausible that alpha-synuclein toxicity is dependent on the phosphorylation at Ser(129) that induces the UPRs, possibly triggered by the disturbed endoplasmic reticulum-Golgi trafficking.