We have examined the chromatin structure of the centromere regions of chromosomes III and XI in yeast by using cloned functional centromere DNAs (CEN3 and CEN11) as labeled probes. When chromatin from isolated nuclei is digested with micrococcal nuclease and the resulting DNA fragments separated electrophoretically and blotted to nitrocellulose filters, the centromeric nucleosomal subunits are resolved into significantly more distinct ladders than are those from the bulk of the chromatin. A discrete protected region of 220-250 bp of CEN sequence flanked by highly nuclease-sensitive sites was revealed by mapping the exact nuclease cleavage sites within the centromeric chromatin. On both sides of this protected region, highly phased and specific nuclease cutting sites exist at nucleosomal intervals (160 bp) for a total length of 12-15 nucleosomal subunits. The central protected region in the chromatin of both centromeres spans the 130 bp segment that exhibits the highest degree of sequence homology (71%) between functional CEN3 and CEN11 DNAs. This unique chromatin structure is maintained on CEN sequences introduced into yeast on autonomously replicating plasmids, but is not propagated through foreign DNA sequences flanking the inserted yeast DNA.