6-Hydroxydopamine (6-OHDA) is widely used to produce animal models of Parkinson's disease (PD) by selectively destroying the nigro-striatal dopaminergic systems, but selective toxicity of 6-OHDA towards dopaminergic cells in vitro remains controversial. Mutant (A30P and A53T) alpha-synuclein isoforms cause increased vulnerability of cells towards various toxic insults and enhance dopamine transporter (DAT)-mediated toxicity of the selective dopaminergic neurotoxin and mitochondrial complex I inhibitor MPP(+) in vitro. Here we extend our recent studies on DAT-mediated toxicity to elucidate the mechanisms involved in selective dopaminergic toxicity of 6-OHDA. We studied the cytotoxicity as well as the toxic mechanisms of 6-OHDA in human embryonic kidney HEK-293 cells ectopically co-expressing mutant alpha-synucleins and the human DAT protein. 6-OHDA showed half-maximal toxic concentration (TC(50)) of 88 microM in HEK-hDAT cells without alpha-synuclein expression after 24 h, whereas the TC(50) values significantly decreased to 58 and 39 microM by expression of A30P and A53T alpha-synuclein, respectively. alpha-Synuclein expression did not affect 6-OHDA toxicity in HEK-293 cells not expressing the DAT. Analysis of intracellular parameters of cellular energy metabolism revealed that the co-expression of mutant alpha-synucleins in HEK-hDAT cells accelerates the reduction of intracellular net ATP levels and ATP/ADP ratios induced by 6-OHDA. Uptake function of the DAT was not altered by expression of alpha-synuclein isoforms. Our data suggest a mechanism of 6-OHDA-induced dopaminergic toxicity involving an interaction of mutant alpha-synucleins with the DAT molecule and subsequent acceleration of cellular energy depletion that might be relevant for the pathogenesis of PD.