Prephosphorylation of τ by cAMP-dependent protein kinase (PKA) significantly stimulated its consecutive phosphorylation catalyzed by glycogen synthetase kinase-3 (GSK-3). After digestion of phosphorylated τ with trypsin, ³²P-labeled τ peptides were purified by a sequential FeCl₃ micro affinity column and a C₁₈ reverse phase high performance liquid chromatography. The results from a combined techniques of high voltage electrophoresis, manual Edman degration and auto gas phase amino acid sequence analysis demonstrated that the phosphorylation sites of τ (pretreated with PKA) by GSK-3 were Ser(serine)-195,Ser-198,Ser-199,Ser-202,Ser-235,Ser-262,Ser-356,Ser-404, Thr(threonine)-205 and Thr-231. Among them, Ser-198,Ser-199,Ser-202,Ser-235,Ser-262,Ser-404,Thr-205 and Thr-231 are abnormal phosphorylation sites of τ found in Alzheimer disease. As the above phosphorylation potently inhibited the biological activity of τ, it was concluded that the phosphorylation of τ at above mentioned Alzheimer sites is critical to the inhibition of its biofunction, and that the interaction of PKA and GSK-3 might be a potential system responsible for the neurofibrillary degeneration seen in Alzheimer disease.