Previous comparisons of centromeric DNA sequences in laboratory strains of Saccharomyces cerevisiae have revealed conserved sequences within 120 base pairs (bp) which appear to be essential for centromere function. We wanted to find out whether centromeric DNA in Saccharomyces strains with different degrees of DNA sequence divergence carry the same conserved sequences or not. Bam HI DNA fragments from two S. cerevisiae strains and one Saccharomyces uvarum strain were cloned into a centromere selection vector and tested for centromere function in a S. cerevisiae laboratory strain. Fragments having centromere function were obtained at approximately equal frequencies from all three strains. Two of the S. uvarum centromeric DNAs and two of the S. cerevisiae centromeric DNAs were sequenced and shown to carry in a 120 bp region sequences essentially like those of centromeric DNA in S. cerevisiae laboratory strains. DNA hybridization to separated chromosomal DNAs revealed that the two newly determined S. cerevisiae centromeric DNA sequences belong to chromosomes V and XIII, respectively.