Tau is a microtubule associated protein in neuron. The biological functions of tau are to stimulate microtubule assembly and to stabilize microtubule structure. These functions are regulated by its phosphorylation status. Abnormally phosphorylated tau is a major component of neurofibrillary tangles. As one of the major tau kinases, glycogen synthase kinase-3β (GSK-3β) can phosphorylate tau at several sites. Site-specific phosphorylation of tau by GSK-3β and the kinetics of GSK-3β on tau phosphorylation was investigated. Tau₄₄₁ was phosphorylated by recombinant GSK-3β in vitro, and the site-specific phosphorylation was detected by Western blots using site-specific and phosphorylation-dependent tau antibodies. The kinetics of phosphorylation by GSK-3β to total tau and to individual site was studied by incubating GSK-3β with various concentration of tau at 30℃ for 10 min. These velocities of phosphorylation reaction were determined at various concentrations of tau by measuring ₃₂P incorporation to tau and phosphorylation level at individual site detected with immuno-dot blots using site-specific and phosphorylation dependent anti-tau antibodies, respectively. The K_m values of GSK-3β toward total tau and toward individual site were calculated by using Lineweaver-Burk double-reciprocal method. The site-specific phosphorylation of tau by GSK-3β was further confirmed in cultured CHO cells by co-transfection of tau₄₄₁ with GSK-3β. It was found that GSK-3β phosphorylated tau at several sites, including Thr181, Ser199, Ser202, Thr205, Thr212, Thr 217, Thr231, Ser396, and Ser404. The Km value of GSK-3β toward total tau was 42 μmol/L, but the K_m value of GSK-3β to every site was different. Among all the phosphorylation sites detected here, Ser396 phosphorylation catalyzed by GSK-3β showed the lowest K_m, only 16 μmol/L. In cultured CHO cells, GSK-3β over-expression also induced tau phosphorylation at Ser396 the most dramatically. These results suggested that GSK-3β catalyzed tau phosphorylation at several sites with different efficiency, and Ser396 was the most efficient site for phosphorylation by GSK-3β .