The epitope on tau protein recognized by the monoclonal antibody Alz50 was defined through internal deletion mutagenesis and quantified by affinity measurements. The epitope is discontinuous and requires both a previously identified N-terminal segment and the microtubule binding region for efficient binding of Alz50. The interaction between these regions is consistent with an intramolecular reaction mechanism, suggesting that Alz50 binding depends on the conformation of individual tau monomers. The results suggest that tau adopts a distinct conformation when polymerized into filaments and that this conformation is recognized selectively by Alz50.