Microtubule-associated protein tau is abnormally hyperphosphorylated and aggregated into neurofibrillary tangles in brains with Alzheimer's disease. The phosphorylation sites of tau are mainly localized in the proline-rich (residues 172-251) and C-terminal tail (residues 368-441) regions, which flank the microtubule-binding repeats. Here, we investigated the effects of tau phosphorylation at these distinct sites/regions on its activity of stimulating microtubule assembly and its self-aggregation. We found that tau phosphorylation at the proline-rich region by dual-specificity tyrosine-phosphorylated and -regulated kinase 1A inhibited its microtubule assembly activity moderately and promoted its self-aggregation slightly. Tau phosphorylation at the C-terminal tail region by glycogen synthase kinase-3beta increased its activity and promoted its self-aggregation markedly. Tau phosphorylation at both regions plus the microtubule-binding region by cAMP-dependent protein kinase diminished its activity (approximately 70% inhibition) and disrupted microtubules. These studies reveal the differential regulation of tau's biological activity and self-aggregation by phosphorylation at various sites/regions.