Human centromere formation involves the assembly of the mitotic kinetochore onto chromosomal locations that contain the interphase prekinetochore. Immunofluorescent analysis of two functionally converse human centromere variants, neocentromeres and inactive centromeres, has been used to evaluate the functional significance of over 24 CENTROMERE proteins, providing important insight into the epigenetics of centromere formation and kinetochore assembly.