This is the author accepted manuscript. The final version is available from Elsevier via http://dx.doi.org/10.1016/j.jmb.2016.04.002 ; Human ubiquitin C-terminal hydrolase, UCH-L1, is an abundant neuronal deubiquitinase that is associated with Parkinson?s disease. It contains a complex Gordian knot topology formed by the polypeptide chain alone. Using a combination of fluorescence-based kinetic measurements, we show that UCH-L1 has two distinct kinetic folding intermediates which are transiently populated on parallel pathways between the denatured and native states. NMR hydrogendeuterium exchange (HDX) experiments indicate the presence of partially unfolded forms (PUFs) of UCH-L1 under native conditions. HDX measurements as a function of urea concentration were used to establish the structure of the PUFs and pulsed-label HDX NMR was used to show that the PUFs and the folding intermediates are likely the same species. In both cases, a similar stable core encompassing most of the central ?-sheet is highly structured and ?-helix 3, which is partially formed, packs against it. In contrast to the stable ?-sheet core, the peripheral ?-helices display significant local fluctuations leading to rapid exchange. The results also suggest that the main difference between the two kinetic intermediates is structure and packing of ?-helices 3 and 7 and the degree of structure in ?-strand 5. Together, the fluorescence and NMR results establish that UCH-L1 neither folds through a continuum of pathways nor by a single discrete pathway. Its folding is complex, the ?-sheet core forms early and is present in both intermediate states, and the rate-limiting step which is likely to involve the threading of the chain to form the 5_2-knot occurs late on the folding pathway. ; This work was supported by the National Science Council (99-2911-I-007-034 and 104-2113-M-001-016), National Tsing Hua University and Academia Sinica, Taiwan. S.-T.D.H. was supported by a Career Development Award (CDA- 00025/2010-C) from the International Human Frontier Science Program. The NMR spectra were obtained at the NMR facility of the Department of Chemistry, University of Cambridge and at the Core Facility for Protein Structural Analysis, supported by the National Core Facility Program for Biotechnology, Taiwan.