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The Ace2p and Swi5p zinc finger proteins have nearly identical DNA-binding domains, yet in vivo they activate transcription of different genes, CTS1 and HO. We now demonstrate that Ace2p and Swi5p recognize sites in the CTS1 and HO promoters with the same affinities, raising the question of how promoter specificity is achieved by these proteins with similar DNA-binding domains. It has been previously shown that Swi5p binds to the HO promoter cooperatively with the Pho2p (Base2p/Grf10p) homeodomain protein, and we now show that Ace2p does not interact with Pho2p. Analysis of CTS1 promoter fragments inserted into a heterologous promoter identify a sequence 90 bp away from the Ace2p binding sites which is required to prevent activation by Swi5p through these binding sites. These results suggest that a regulatory protein bound to the CTS1 promoter is needed to prevent Swi5p from activating CT1S expression. A genetic screen was conducted to identify suppressor mutations which allow CTS1 expression in the absence of the Ace2p activator. The nce3 mutation suppresses the ace2 defect in CTS1 expression only if the strain contains a functional SWI5 gene, suggesting that NCE3 normally functions to prevent Swi5p from activating CTS1. The role of negative regulators such as NCE3, as well as the previously described SIN5 gene, in determining the promoter specificity of homologous activators is discussed.