Cell type in Saccharomyces yeasts is regulated by two transposable blocks of DNA, the a and alpha cassettes. There are three loci where either cassette can exist. At the HML and HMR loci the cassettes are not expressed. The cassette at the MAT locus is expressed and controls the cell type. Changes of cell type involve transposition-substitution of cassettes from HML or HMR into MAT. We recently reported the molecular cloning of the alpha cassette at the HML locus, HML alpha, and showed that it contained sequences homologous to HMR and MAT. Using HML alpha as a hybridization probe, we have isolated HMLa, HMR alpha, HMRa, MAT alpha, and MATa. Heteroduplex analysis and restriction endonuclease mapping studies indicate that the a and alpha cassettes differ by a substitution corresponding to about 750 base pairs in alpha and about 600 base pairs in a. The HML, HMR, and MAT loci have regions of homology flanking the position of the a versus alpha substitution. We have used specific chromosome rearrangements fusing MAT and HML and MAT with HMR to orient the cloned sequences on the genetic map and have found that all three genes have the same left-to-right polarity on the chromosome.