Disorder in sister chromatid separation can lead to genome instability and cancer. A temperature-sensitive S. pombe mis6-302 frequently loses a minichromosome at 26 degrees C and abolishes equal segregation of regular chromosomes at 36 degrees C. The mis6+ gene is essential for viability, and its deletion results in missegregation identical to mis6-302. Mis6 acts before or at the onset of S phase, and mitotic missegregation defects are produced only after the passage of G1/S at 36 degrees C. Mis6 locates at the centromeres throughout the cell cycle. In the mutant, positioning of the centromeres becomes abnormal, and specialized chromatin in the inner centromeres, which give the smear micrococcal nuclease pattern in wild type, is disrupted. The ability to establish correct biorientation of sister centromeres in metaphase cells requires the Mis6-containing chromatin and originates during the passage of G1/S.