Alz‐50 and MC‐1 monoclonal antibody reactivity is dependent on both the extreme N‐terminus of tau (residues 7–9) and a 30–amino acid sequence of tau (amino acids 312–342) in the third microtubule binding domain, suggesting that the specificity of the Alz‐50 and MC‐1 antibodies for Alzheimer's disease (AD) pathological tau lies in their ability to recognize a specific conformation of the tau molecule in AD. The present study uses deletional and site‐directed mutants of tau to further refine the C‐terminal (third microtubule binding domain) epitope requirements for Alz‐50, MC‐1, and several new antibodies that recognize similar epitopes in tau to amino acids 313–322 of tau, and to demonstrate that intervening portions of the tau molecule are not required for the formation of conformational variants of tau similar to those seen in AD. Further analysis of deletional and site‐directed mutations of tau demonstrate subtle variations in the epitope requirements for Alz‐50, MC‐1, CP‐1, CP‐2, and CP‐28, suggesting that these antibodies, albeit different, all recognize a similar pathological conformation of tau. Additional experiments using synthetic peptides demonstrate that the NH₂‐terminal (amino acids 1–18) and COOH‐terminal (amino acids 309–326) portions of the Alz‐50, MC‐1, CP‐1, CP‐2, and CP‐28 epitopes can interact with high affinity under near physiological conditions. J. Neurosci. Res. 56:713–723, 1999. © 1999 Wiley‐Liss, Inc.