A synthetic peptide corresponding to residues 226-240 (E9 peptide) of human tau, which contains an Lys-Ser-Pro motif, was used to raise a polyclonal antibody. The antibody, E9, was 10-fold less reactive with phospho-E9 peptide than with native E9 peptide. E9 antibody was used to study the extent of phosphorylation in a modified form of tau (PHF-tau) that is found in Alzheimer's disease brain and is incorporated into paired helical filaments (PHFs). E9 immunolabeled Alzheimer's disease neurofibrillary tangles and abnormal neurites in brain sections intensely, with increased immunoreactivity detected after pretreatment of sections with phosphatase. On immunoblots and ELISA, E9 reacted with PHF-tau and recombinant human tau but not with the high and middle molecular weight neurofilament proteins. Phosphatase treatment of PHF-tau improved the E9 immunoreactivity by 30-50%. Dephosphorylated high but not middle molecular weight neurofilament protein became reactive with E9. These results indicate that < 50% of the PHF-tau is phosphorylated in the subregion corresponding to residues 226-240 of tau and suggest that the phosphorylation of this region may not be essential for PHF formation.