Alpha-synuclein (α-SYN) is a small and highly conserved presynaptic protein that is implicated in both normal brain function and the pathogenesis of neurodegenerative diseases. Although the physiological function of α-SYN has not been fully evaluated, several lines of evidence suggest that it plays an important role in the regulation of dopamine homeostasis in the brain. Early studies have demonstrated that interaction of α-SYN and tyrosine hydroxylase (TH), a rate-limiting enzyme in dopamine synthesis, is functionally significant for dopaminergic neurotransmission and the pathophysiology of Parkinson's disease. In the present study, we would like to evaluate whether overexpression of wild-type or mutant α-SYN might affect cAMP/PKA-dependent TH activation in DA-producing SK-N-BE(2)C cells. Here we show that wild-type and mutant A30P and A53T α-SYN attenuate forskolin-induced TH up-regulation, but do not suppress TH basal expression in SK-N-BE(2)C cells. Forskolin-induced increase in TH promoter activity and CRE-dependent transcription are significantly suppressed in α-SYN-overexpressing cells. Alpha-SYN enters the nucleus, but does not bind to CREB or interfere with forskolin-induced CREB phosphorylation. These data indicate that elevated levels of α-SYN due to a specific disease or the normal aging process could be associated with dopaminergic neuronal dysfunction through interference with TH regulation.