Nucleosome positioning has been proposed as a mechanism of transcriptional repression. Here, we examined whether nucleosome positioning affects activator binding in living yeast cells. We introduced the cognate Hap1 binding site (UAS1) at a location 24-43 bp, 29-48 bp, or 61-80 bp interior to the edge of a nucleosome positioned by alpha2/Mcm1 in yeast minichromosomes. Hap1 binding to the UAS1 was severely inhibited, not only at the pseudo-dyad but also in the peripheral region of the positioned nucleosome in alpha cells, while it was detectable in a cells, in which the nucleosomes were not positioned. Hap1 binding was restored in alpha cells with tup1 or isw2 mutations, which caused the loss of nucleosome positioning. These results support the mechanism in which alpha2/Mcm1-dependent nucleosome positioning has a regulatory function to limit the access of transcription factors.