THE microtubule-associated protein tau is abnormally hyperphosphorylated in Alzheimer's disease (AD) brain. To date, 21 phosphorylated sites of tau have been identified. In the present study the levels of phosphorylation at Ser199/Ser202, Thr231/Ser235, Ser262/Ser356and Ser396/Ser404of tau in AD brain homogenate and its 100 000 × g supernatant were determined using radioimmuno-dot-blot assay. In homogenate, Ser199/Ser202and Ser262/Ser356were phosphorylated to similar level and were more phosphorylated than Thr231or Ser396/Ser404. In supernatant, there was no significant difference in phosphorylated tau level among the investigated sites except for Thr231/Ser235which was least phosphorylated. These results suggest that Ser199/Ser202and Ser262/Ser356are major sites of phosphorylation of tau in AD brain.