It has been proposed that yeast AL4Ta cell-specific genes are repressed in MATa cells by the Mata2p repressor-directed placement of a nucleosome in a position that incorporates the TATA box of the AMTa-specific gene close to the nucleosomal pseudodyad. In this study, we address this proposal directly with a series of plasmids designed to place the MATa-specific STE6 TATA box at different locations in a nucleosome and in the internucleosomal linker. These plasmids contain different lengths of synthetic random DNA between the Mata2p operator and the TATA box of the STE6 promoter, which is located upstream of a lacZ reporter gene in a multicopy plasmid. We proposed that the role of Mata2p is to recruit the Ssn6p-Tuplp complex which in turn mediates repression of the AMTaspecific gene (24). Although several mechanisms for Mata2p-mediated AMTa-specific gene repression have been proposed (15,21,23,44,45,48), the molecular basis of this repression is not understood.The most striking structural feature of a repressed AL4Ta-specific gene in a AM Ta cell is the presence of a regular array of nucleosomes covering the promoter area and extending into the structural gene (48). The generation of this Mata2p-dependent nucleosome array adjacent to the Mata2p operator has been characterized in the TRPlARS1-derived multicopy TASTE and TALS plasmids (44), as well as in the single-copy genomic AMTa-specific STE6 and BAR1 genes (48). In the TALS plasmid, two nucleosomes were shown to resolve in precise translational frames (44) and to occupy a dominant rotational frame (48)