AbstractTangles containing hyperphosphorylated aggregates of insoluble tau are a pathological hallmark of progressive supranuclear palsy (PSP). Several phosphorylation sites on tau in PSP have been identified using phospho‐specific antibodies, but no sites have been determined by direct sequencing due to the difficulty in enriching insoluble tau from PSP brain. We describe a new method to enrich insoluble PSP‐tau and report eight phosphorylation sites [Ser46, Thr181, Ser202, Thr217, Thr231, Ser235, Ser396/Ser400 (one site) and Thr403/Ser404 (one site)] identified by mass spectrometry. We also describe a 35 kDa C‐terminal tau fragment (tau35), lacking the N‐terminus of tau but containing four microtubule‐binding repeats (4R), that is present only in neurodegenerative disorders in which 4R tau is over‐represented. Tau35 was readily detectable in PSP, corticobasal degeneration and 4R forms of fronto‐temporal dementia with parkinsonism linked to chromosome 17, but was absent from control, Alzheimer’s disease and Pick’s disease brain. Our findings suggest the aggregatory characteristics of PSP‐tau differ from those of insoluble tau in Alzheimer’s disease brain and this might be related to the presence of a C‐terminal cleavage product of tau.