Lewy bodies are found in Parkinson disease and related disorders and are extensively phosphorylated at Ser-129 (S129), but whether S129 phosphorylation mediates alpha-synuclein aggregation and neurotoxicity has been controversial. We used recombinant adeno-associated virus to overexpress alpha-synuclein in the rat nigrostriatal system. Rats were injected with recombinant adeno-associated virus 2/8 expressing either human wild-type (wt) or mutant alpha-synuclein with S129 replaced by alanine (S129A) or aspartate (S129D). Contralateral substantia nigra injections containing empty vector served as controls. Both wt and S129 mutants resulted in significant dopaminergic cell loss in the recipients by 6 weeks, but there were only small decreases in nigrostriatal terminal density and tyrosine hydroxylase expression. There were no significant differences in dopaminergic cell loss, nigrostriatal terminal density, or tyrosine hydroxylase expression among the wt and S129 mutants. Furthermore, we did not observe any differences in alpha-synuclein aggregate formation or distribution among wt and either S129 mutant. These findings contrast with those from previous studies and suggest that injections of both S129 phosphorylation mutants result in dopaminergic neurotoxicity similar to wt injections. Further study is needed to clarify the effects of these S129 mutants and alpha-synuclein phosphorylation in mammalian systems.