We determined the nucleotide sequence of DNA segments containing functional centromeres (CEN3 and CEN11) isolated from yeast chromosomes III and XI. The two centromere regions differ in primary nucleotide sequence, but contain structural features in common. Both centromere regions contain an extremely A + T-rich core segment 87-88 bp in length, flanked by two short sequences (14 bp and 11 bp) that are identical in both DNAs. These elements plus one additional 10 bp region of perfect homology are positioned in an almost identical spatial arrangement within the two centromere regions. Significant homologies are also observed among the sequences flanking the high A + T region and various satellite DNA sequences from higher eucaryotes, although no repeated sequences occur near the yeast centromeres. Centromere activity in vivo is maintained on relatively small DNA fragments (627 bp for CEN3 and 858 bp for CEN11), as assayed by mitotic stabilization of autonomously replicating ars plasmids in yeast.