BACKGROUND: Sister kinetochores are bioriented toward the spindle poles in eukaryotic metaphase before chromosome segregation. In the budding yeast Saccharomyces cerevisiae, sister centromeres/kinetochores are separated in the early spindle, while the sister arms remain associated. Biorientation is thought to be established in this organism with precocious separation of sister centromeres in early stages of the cell cycle. It is not, however, settled whether this pre-anaphase separation is continuous or only transient and whether the transient separation has any physiological significance. RESULTS: Time-lapse observation of the behaviour of budding yeast centromeres in living cells was performed using GFP alone or in combination with CFP marking. Sixty-three per cent of the cell population showed permanent separation of centromeres for a long period of time from the small-budded stage to the onset of anaphase in the single-colour GFP-CEN construct. The remaining cell population (6 of 16) showed brief apparent reassociation of centromere signals before anaphase, but the frequency of the association was very low. In a time-lapse observation of the double-colour marked cells by GFP-CEN and CFP-SPB (the spindle pole body), the continuous separation of sister centromeres in the short medial spindle was firmly established. CONCLUSIONS: In the budding yeast, once sister centromeres separate, they rarely reassociate in pre-anaphase. Sister centromere cohesion at this stage appears to be irrelevant for normal chromosome segregation. Whether abundant cohesin in the centromere regions has any role in anaphase remains to be determined.