Huntington's disease is a dominantly inherited, devastating neurodegenerative disorder, caused by a polyglutamine expansion (>37) in the N-terminal region of huntingtin, a protein of unknown function. In patients and normal individuals, N-terminal fragments of huntingtin are found, and the N-terminal fragments of mutant huntingtin are cytotoxic. The functions of wild-type huntingtin and the mechanisms underlying the toxic effects of mutant huntingtin are still ill defined. To get more insight into these topics, monoclonal antibodies (MAbs) are indispensable tools. Antibodies raised against the N-terminus are especially important. Among these, the 4C8 mouse MAb has been extensively used in various approaches. In this study, we have mapped the epitope of 4C8 to a 15-amino acid (aa) region spanning from aa 443 to 457 of the human protein, and found that mutation of three consecutive glutamic acids present in this region disrupts the recognition by 4C8. These results allow a more accurate interpretation of the results obtained by usage of the 4C8 antibody and broaden the utility of this antibody.