BACKGROUND Precise control of sister chromatid separation is essential for the accurate transmission of genetic information. Sister chromatids must remain linked to each other from the time of DNA replication until the onset of chromosome segregation, when the linkage must be promptly dissolved. Recent studies suggest that the machinery that is responsible for the destruction of mitotic cyclins also degrades proteins that play a role in maintaining sister chromatid linkage, and that this machinery is regulated by the spindle-assembly checkpoint. Studies on these problems in budding yeast are hampered by the inability to resolve its chromosomes by light or electron microscopy. RESULTS We have developed a novel method for visualizing specific DNA sequences in fixed and living budding yeast cells. A tandem array of 256 copies of the Lac operator is integrated at the desired site in the genome and detected by the binding of a green fluorescent protein (GFP)-Lac repressor fusion expressed from the HIS3 promoter. Using this method, we show that sister chromatid segregation precedes the destruction of cyclin B. In mad or bub cells, which lack the spindle-assembly checkpoint, sister chromatid separation can occur in the absence of microtubules. The expression of a tetramerizing form of the GFP-Lac repressor, which can bind Lac operators on two different DNA molecules, can hold sister chromatids together under conditions in which they would normally separate. CONCLUSIONS We conclude that sister chromatid separation in budding yeast can occur in the absence of microtubule-dependent forces, and that protein complexes that can bind two different DNA molecules are capable of holding sister chromatids together.