AbstractParkinson's disease (PD) is characterized by the accumulation of misfolded alpha‐synuclein (α‐syn) protein, forming intraneuronal Lewy body (LB) inclusions. The α‐syn preformed fibril (PFF) model of PD recapitulates α‐syn aggregation, progressive nigrostriatal degeneration and motor dysfunction; however, little is known about the time course of PFF‐induced alterations in basal and evoked dopamine (DA). In vivo microdialysis is well suited for identifying small changes in neurotransmitter levels over extended periods. In the present study, adult male Fischer 344 rats received unilateral, intrastriatal injections of either α‐syn PFFs or phosphate‐buffered saline (PBS). At 4 or 8 months post‐injection (p.i.), animals underwent in vivo microdialysis to evaluate basal extracellular striatal DA and metabolite levels, local KCl‐evoked striatal DA release and the effects of systemic levodopa (l‐DOPA). Post‐mortem analysis demonstrated equivalent PFF‐induced reductions in tyrosine hydroxylase (TH) immunoreactive nigral neurons (~50%) and striatal TH (~20%) at both time points. Compared with reduction in striatal TH, reduction in striatal dopamine transporter (DAT) was more pronounced and progressed between the 4‐ and 8‐month p.i. intervals (36% ➔ 46%). Significant PFF‐induced deficits in basal and evoked striatal DA, as well as deficits in motor performance, were not observed until 8 months p.i. Responses to l‐DOPA did not differ regardless of PBS or PFF treatment. These results suggest that basal and evoked striatal DA are maintained for several months following PFF injection, with loss of both associated with motor dysfunction. Our studies provide insight into the time course and magnitude of PFF‐induced extracellular dopaminergic deficits in the striatum.