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5-Bromodeoxyuridine (BrdU) modulates the expression of particular genes associated with cellular differentiation and senescence when incorporated into DNA instead of thymidine (dThd). To date, a molecular mechanism for this phenomenon remains a mystery in spite of a large number of studies. Recently, we have demonstrated that BrdU disrupts nucleosome positioning on model plasmids mediated by specific AT-tracts in yeast cells. Here we constructed a cognate plasmid that can form an ordered array of nucleosomes determined by an α2 operator and contains the BAR1 gene as an expression marker gene to examine BAR1 expression in dThd-auxotrophic MATα cells under various conditions. In medium containing dThd, BAR1 expression was completely repressed, associated with the formation of the stable array of nucleosomes. Insertion of AT-tracts into a site of the promoter region slightly increased BAR1 expression and slightly destabilized nucleosome positioning dependent on their sequence specificity. In medium containing BrdU, BAR1 expression was further enhanced, associated with more marked disruption of nucleosome positioning on the promoter region. Disruption of nucleosome positioning seems to be sufficient for full expression of the marker gene if necessary transcription factors are supplied. Incorporation of 5-bromouracil into the plasmid did not weaken the binding of the α2/Mcm1 repressor complex to its legitimate binding site, as revealed by an in vivo UV photofootprinting assay. These results suggest that BrdU increases transcription of repressed genes by disruption of nucleosome positioning around their promoters.