OBJECTIVE: The objective of this study was to provide clinical and pathological characterization, and evaluation of the diagnostic and prognostic implications of levodopa response in Parkinson's disease (PD), multiple system atrophy (MSA) and progressive supranuclear palsy (PSP). METHODS: Long-duration levodopa response in patients with pathology-confirmed PD, MSA, and PSP was collated from medical records. Associations between definite levodopa response (>50% motor improvement sustained >2 years) with clinical and pathological features were reported. Risk of disease milestones using multivariate Cox regression and diagnostic accuracy parameters were estimated for several measures of levodopa response. RESULTS: Fourteen percent of 132 patients with PD (57.1% men, age at onset 60.0 ± 11.8 years) did not have a definite levodopa response associated with older age and postural-instability and gait-difficulty subtype without global or nigral pathological differences. Definite levodopa responders had 55% lower falls, 69% lower dementia risk, and 69% increased survival. Eight percent of 115 patients with MSA (55.7% men, age at onset 57.9 ± 10.4 years) showed a definite levodopa response without distinctive clinical or pathological features, and no impact on disease milestones. Two percent of 191 patients with PSP (64.4% men, age of onset 67.6 ± 7.6 years) showed a definite levodopa response associated with a PSP-parkinsonism subtype, and more frequent and severe Lewy copathology. Definite levodopa response showed excellent diagnostic accuracy (sensitivity 86.4% and specificity 95.8%) to distinguish PD from MSA and PSP. Short-duration (acute dopaminergic drug challenge) response had suboptimal diagnostic value and modest correlation with long-duration response. INTERPRETATION: Levodopa response in PD, MSA, and PSP has important clinical, pathological, diagnostic, and prognostic implications. ANN NEUROL 2026.