The nucleotide sequences of five of the six centromeres of the yeast Kluyveromyces lactis were determined. Mutual comparison of these sequences led to the following consensus: a short highly conserved box (5'-ATCACGTGA-3') flanked by an AT-rich (+/- 90%) stretch of +/- 160 bp followed by another conserved box (5'-TNNTTTATGTTTCCGAAAATTAATAT-3'). These three elements were named KlCDEI, KlCDEII, and KlCDEIII respectively, by analogy with the situation in Saccharomyces cerevisiae. In addition, a second 100 bp AT-rich (+/- 90%) element, named KlCDE0, was found +/- 150 bp upstream of KlCDEI. The sequences of both KlCDEI and KlCDEIII are highly conserved between K. lactis and S. cerevisiae; however, centromeres of K. lactis do not function in S. cerevisiae and vice versa. The most obvious differences between the centromeres of the two yeast species are the length of the AT-rich CDEII, which is 161-164 bp in K. lactis versus 78-86 bp in S. cerevisiae and the presence in K. lactis of KlCDE0, which is not found in S. cerevisiae.