Alpha-synuclein (α-syn) is an abundant presynaptic protein that associates with the surface of synaptic vesicles (SVs). It is the main component of Lewy bodies, abnormal protein aggregates that serve as a hallmark of both sporadic and familial forms of Parkinson's disease (PD). While its physiological role in the synapse remains elusive, recent studies indicate it attenuates synaptic transmission and regulates SV clustering. Specifically, when human α-syn (h-α-syn) is over-expressed in mouse neurons by viral transduction or in transgenic mice, it inhibits SV recycling and disperses the SV clusters. Although h-α-syn and its murine ortholog (m-α-syn) differ significantly at the mRNA level, at the protein level they are 95% identical, differing by only 7/140 amino acids. We found that, unlike h-α-syn, m-α-syn does not attenuate SV recycling, as measured using synaptophysin-pHluorin, a sensor of exocytosis/endocytosis. Furthermore, by analyzing the presynaptic distribution and density of both orthologs, as well as of vGlut1, an SV marker in glutamatergic neurons, we found that over-expression of h-α-syn disperses the SV clusters, but a comparable expression of m-α-syn does not. Concomitantly, m-α-syn localizes to the presynaptic terminal to a higher degree. To explore the molecular basis for the functional differences between the orthologs, we tested the synaptic effect of a major PD-related mutation in h-α-syn (A53T) which alters the human sequence to the murine one. This mutant maintains the capability of the human ortholog to attenuate SV recycling, indicating that one or more of the other alterations between the orthologs are responsible for their functional differences.