Alzheimer's disease and other tauopathies are characterized by the brain accumulation of hyperphosphorylated aggregated tau protein forming pathological inclusions. Although elevated tau phosphorylated at many amino acid residues is a hallmark of pathological tau， some evidence suggest that tau phosphorylation at unique sites， especially within its microtubule-binding domain， might inhibit aggregation. In this study， the effects of phosphorylation of two unique residues within this domain， serine 305 (S305) and serine 320 (S320)， were examined in the context of established aggregation and seeding models. It was found that the S305E phosphomimetic significantly inhibited both tau seeding and tau aggregation in this model， while S320E did not. To further explore S305 phosphorylation in vivo， a monoclonal antibody (2G2) specific for tau phosphorylated at S305 was generated and characterized. Consistent with inhibition of tau aggregation， phosphorylation of S305 was not detected in pathological tau inclusions in Alzheimer's disease brain tissue. This study indicates that phosphorylation of unique tau residues can be inhibitory to aggregate formation， and has important implications for potential kinase therapies. Additionally， it creates new tools for observing these changes in vivo.