Numerous studies suggest that dysfunction of mitochondrial proton-translocating NADH-ubiquinone oxidoreductase (complex I) is associated with neurodegenerative disorders, such as Parkinson's disease and Huntington's disease. Development of methods to correct complex I defects seems important. We have previously shown that the single-subunit NADH dehydrogenase of Saccharomyces cerevisiae (Ndi1P) can work as a replacement for complex I in mammalian cells. Using a recombinant adeno-associated virus vector carrying the NDI1 gene, we now demonstrated that the Ndi1 enzyme was successfully expressed in the dopaminergic cell lines rat PC12 and mouse MN9D. The cells expressing the Ndi1 protein were resistant to known inhibitors of complex I, such as rotenone and pyridaben. In addition, the NDI1-transduced cells were still capable of morphological maturation as examined by induction of neurite outgrowth. Also, it was possible to infect the cells after the maturation. The expressed Ndi1 protein was located both in cell bodies and in neurites and was functionally active. It is conceivable that the NDI1 gene will be a promising tool in the treatment of neurodegenerative conditions caused by complex I inhibition.