Whereas the major satellite fraction in mouse extends its domain from the centromere to the distal end of the pericentric heterochromatin, the minor satellite DNA is present specifically in the centromere or primary constriction. We hybridized the biotinylated minor satellite sequence to L929 cells of mouse origin. The sequence hybridized to all chromosomes. Whereas hybridization was detected on all active centromeres, the inactive centromeres in certain dicentrics did not show any signal. This satellite, however, was detected in all inactive centromeres in a heptacentric chromosome. The intensity of fluorescence on the inactive centromeres of the heptacentric was similar to that present on the active centromeres. Several heterochromatin blocks, which were not associated with any centromere, were also found to lack hybridization with the minor satellite. The inactive centromeres, whether carrying the minor satellite DNA fraction or not, generally do not react with the antikinetochore antibodies present in the scleroderma serum. These studies are interpreted to show that (1) the primary constriction in mouse can be formed without the participation of minor satellite, (2) heterochromatin in mouse may constitute without this fraction, (3) the major and minor satellite may not be interspersed but are joined at some defined boundary, and (4) the binding of CENP-B does not depend upon the quantity of minor satellite or the number of CENP boxes present in the inactive centromeres.