In Saccharomyces cerevisiae, a-cell-specific genes are repressed in MATalpha cells by alpha2/Mcm1, acting in concert with the Ssn6-Tup1 corepressors and the Isw2 chromatin remodeling complex, and nucleosome positioning has been proposed as one mechanism of repression. However, prior studies showed that nucleosome positioning is not essential for repression by alpha2/Mcm1 in artificial reporter plasmids, and the importance of the nucleosome positioning remains questionable. We have tested the function of positioned nucleosomes through alteration of genomic chromatin at the a-cell-specific gene BAR1. We report here that a positioned nucleosome in the BAR1 promoter is disrupted in cis by the insertion of diverse DNA sequences such as poly(dA) . poly(dT) and poly(dC-dG) . poly(dC-dG), leading to inappropriate partial derepression of BAR1. Also, we show that isw2 mutation causes loss of nucleosome positioning in BAR1 in MATalpha cells as well as partial disruption of repression. Thus, nucleosome positioning is required for full repression, but loss of nucleosome positioning is not sufficient to relieve repression completely. Even though disruption of nucleosome positioning by the cis- and trans-acting modulators of chromatin has a modest effect on the level of transcription, it causes significant degradation of the alpha-mating pheromone in MATalpha cells, thereby affecting its cell type identity. Our results illustrate a useful paradigm for analysis of chromatin structural effects at genomic loci.