SWI5 encodes a zinc finger DNA-binding protein required for HO transcription, and PHO2 encodes a homeodomain DNA-binding protein. In vitro studies using purified Swi5p and Pho2p proteins have demonstrated that these proteins bind cooperatively to the HO promoter. Deletion analysis of the proteins also revealed that DNA-binding domains of either were sufficient for binding to the HO promoter but additional regions were required for cooperative binding (JBC 270:29151). Further, changing the spacing between the binding sites does not block cooperative interactions, suggesting flexibility in at least one protein's interaction domain. Finally, Pho2p may interact with other proteins, Pho4p and Bas1p, to activate other genes. The current work was undertaken to explore the interactive surfaces of both proteins. We have developed a screen to identify specific residues of Swi5p required for cooperative interaction with Pho2p. A plasmid library of mutagenized SWI5 was screened for mutants specifically defective in Pho2p interaction. A secondary screen was used to eliminate functionally inactive SWI5 mutants that failed to activate PHO2-independent promoter constructs. Four SWI5 mutants with single amino acid substitutions, all within a 20 amino acid region, have been identified. In vitro DNA-binding studies demonstrate that the DNA-binding ability of these mutants is not affected, but they are defective in cooperative DNA-binding with Pho2p.