Alzheimer's disease (AD) paired helical filaments (PHFs), building blocks of neurofibrillary tangles (NFTs) are composed of hyperphosphorylated forms of the microtubule-associated protein tau (i.e., PHF-tau). Currently, much effort is devoted to the development of diagnostic antibodies specific for PHF-tau since elevated tau levels are found in the cerebral spinal fluid of AD patients. To this end, we have mapped the epitopes of a large panel of monoclonal antibodies (mAbs) that recognized only phosphorylation dependent epitopes on PHF-tau. These mAbs include the PHF-tau specific mAb AT10 and 12 newly developed anti-PHF mAbs that recognize PHF-tau but not autopsy-derived normal adult tau on Western-blot and enzyme-linked immunosorbent assay (ELISA). Epitope analysis, together with data on known binding sites of previously published mAbs, revealed that Ser214, Thr231, and Ser396 are immunodominant phosphorylated amino acids in PHF-tau. Six of the 12 new mAbs recognized one of these three phosphorylated sites. With the exception of AT10 and PHF-27, all the mAbs also labeled fetal tau and biopsy-derived tau. Since mAbs AT10 and PHF-27 had little or no affinity for fetal tau and biopsy tau, they can be considered as the first "true" PHF-specific antibodies capable of distinguishing tau isoforms from normal versus AD subjects, suggesting a possible utility of these mAbs as diagnostic markers. Remarkably, the true PHF-specific antibodies recognized peptide sequences phosphorylated on more than one amino acid residue. The peptide recognition of mAb AT10 required the simultaneous phosphorylation of Thr212 and Ser214, and the peptide recognition of mAb PHF-27 was markedly increased when both the primary site Thr231 and the subsite Ser235 were phosphorylated. Since AT10 and PHF-27 are the only mAbs currently available that bind specifically to PHF-tau, these data suggest that double phosphorylation at Thr212/Ser214 and Thr231/Ser235 may be unique to PHF-tau. These data may facilitate the development of mAbs that can be used as specific diagnostic reagents for the detection of altered tau in cerebrospinal fluid of AD patients.