Antigens associated with mammalian centromeres were localized at the high and electron microscopic levels using the peroxidase-labeled antibody method. The antibody used was of a type naturally occurring in the sera of patients with scleroderma. At the light microscopic level, it reacts specifically with the centromere regions of chromosomes in a variety of mammalian species and strains in discrete foci in interphase nuclei. We find that the number of foci approximates the number of chromosomes present in the various cell types. At the ultrastructural level, the antigenic foci are confirmed to lie in the kinetochore regions of each chromosome. In interphase nuclei, the antigenic foci were usually associated either with the inner surfaces of the nuclear envelope or with the nucleoli. These observations indicate that the centromere regions of the chromosomes in interphase are not randomly distributed within the nucleus but are usually fixed either to the inner surface of the nuclear envelope or to nucleoli.