The 2-microns plasmid of industrial and laboratory strains of Saccharomyces cerevisiae exists as two main polymorphic forms designated type I and type II. Polymorphism is restricted to the 3200-bp right unique region where types I and II show approximately 10% nucleotide divergence in trans-acting REP1 and RAF loci and 30% divergence in the cis-acting STB locus. In addition, the cis-acting STB plasmid partition locus of type II plasmids varies in sequence and copy number of a 125-bp repeat. We devised chimeric and 2-microns plasmid stability experiments to evaluate the effect of STB polymorphism on plasmid fitness in amphiploid industrial and haploid laboratory strains. Reciprocal experiments of type-II STB chimeric plasmids in type-I bakers' yeast or a type-I chimeric plasmid in type-II distillers', wine, or haploid strains showed similar partition efficiencies. However, chimeric and 2-microns plasmids carrying a 250-bp STB from a type-II haploid strain had reduced fitness in a type-II industrial wine strain. These results in conjunction with molecular analyses of 2-microns-like and 2-microns plasmids indicates the coevolution of STB with trans-acting plasmid and host-cell factors.