A temperature-sensitive (ts) mutant of Saccharomyces cerevisiae was isolated in which mini-chromosomes were unstable at high temperature. The MCS1 gene (Mini-Chromosome Stability 1) was cloned by the ability of complementing the temperature sensitivity, and was found to be identical to SSD1/SRK1/SSL1. When MCS1/SSD1 was disrupted in a certain wild-type (wt) strain, mini-chromosomes were unstable, even at 30 degrees C, indicating that the gene is involved in chromosome stability. The Mcs1/Ssd1 protein was detected as a 170-kDa protein by immuno-blotting analysis and this 170-kDa protein could not be detected in the ts mutant and certain wt strains. Our results are consistent with the genetic data that there are two polymorphic forms of the gene, SSD1-v and ssd1-d [Sutton et al., Mol. Cell. Biol. 11 (1991) 2133-2148]. Furthermore, genetic backgrounds other than MCS1/SSD1 caused strain-specific phenotype. The protein, precipitated by specific antibodies, was phosphorylated.