Abstract: The mating-type locus (MAT) of Saccharomyces cerevisiae is a complex locus that codes for the regulators of cell type. Two unique messages are transcribed from each MAT allele. Using the in vitro mutagenesis technique whereby synthetic oligonucleotides containing restriction sites (linkers) were inserted into plasmids, we have constructed a series of mutations in cloned DNA containing either the MATa or MAT alpha locus. The new restriction site associated with each "linker" mutation has allowed the mutation to be mapped and sequenced. We have complemented genetically defined mutations (mata1, mat alpha 1 and mat alpha 2) with plasmids containing these in vitro mutations by yeast transformation, thereby mapping the genes onto the DNA sequence. MATa1 has been localized to the MATa unique region (Ya) from which the a1 message is transcribed. We find no function for the other MATa message by using our complementation assay. MAT alpha 1 maps to the MAT alpha unique (Y alpha) and adjacent (Z) region from which the alpha 1 message is transcribed. MAT alpha 2 maps to the other major message found in the common (X) region of the MAT alpha loci. Although most linker mutations that have a mutant phenotype appear to disrupt the translated portion of each gene, two mutations may disrupt transcription.