Structure-function analysis of a yeast (Saccharomyces cerevisiae) centromere (CEN3) has been carried out by altering the nucleotide sequence of the DNA within and surrounding the centromere of yeast chromosome III, and observing the behaviour of the resulting altered chromosomes during mitotic and meiotic cell divisions. A centromere substitution vector (pJC3-13) was constructed, which contains in the proper orientation: the DNA sequences that normally flank the chromosome III centromere, a wild-type URA3 gene for selection, and a unique BamHI restriction site for insertion of various DNA sequences to be assayed for centromere activity. Cleavage of the plasmid DNA with EcoRI generates a linear DNA fragment whose ends are homologous with the regions flanking the centromere. Transformation of the appropriate homozygous ura3 diploid yeast strain with this linear DNA results in URA3+ transformants in which the CEN3 region on one copy of chromosome III has been replaced by the URA3 gene and the DNA sequence previously inserted into the vector. These studies identify a 289 base-pair (bp) DNA fragment from the CEN3 region that retains full centromere function when used to replace the normal CEN3 sequence. Centromeres function equally well in either orientation, and the chromosome XI centromere (CEN11) can be used to replace CEN3, with no observable effect on mitotic or meiotic chromosome segregation. Various DNA restriction fragments occurring within the CEN3 region were used alone or in combinations to replace the normal CEN3 sequence. Yeast centromeres contain a high A + T region about 82-89 bp in length (element II) flanked by a highly conserved 11 bp sequence (III) and a less-conserved 14 bp sequence (I). The experiments demonstrate that both regions II and III are necessary for normal centromere function, although centromeres containing III plus truncated or rearranged portions of the high A + T region II retain partial activity. Chromosomes of the latter type often give abnormal segregation patterns through meiosis, including separation and random segregation of sister chromatids during the first meiotic division.