The location of histone H5 on nucleosomes has been determined by binding anti-H5 antibodies to dinucleosomes, and recording the position of the bound IgG molecules using electron microscopy. Two types of antibody were employed, a total IgG fraction prepared from rabbits immunized with H5/RNA, which reacted to all three domains (NH2-terminal, central globular, and COOH-terminal) of H5, and an immunospecific subfraction which bound only to the central globular peptide. After reacting with dinucleosomes, both types of antibody were localized primarily in the linker DNA entry/exit region, but the whole antibody showed a much greater affinity for the linker DNA itself than did the antiglobular peptide antibody. These results provide direct support for the concept that H5, and by inference H1, is located at the linker DNA entry/exit site of the nucleosome, and further suggest that it is the central globular portion of the molecule that is most closely associated with this site. An interaction of one or both termini of H5 with the linker DNA is also indicated.