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The yeast two-hybrid system is a useful tool for identifying new protein-protein interactions, and for the dissection of previously identified interactions. An important issue in protein-interaction studies is frequently that of determining whether a protein associates specifically with one protein or domain of interest, or has a more promiscuous interaction profile. To help address this issue, the authors have created a new two-hybrid system, which can be used either in bacteria or in yeast to counterscreen against "decoy" baits in parallel with a primary screen, hence improving the power and specificity of the method. Protocols of this system for use in yeast are provided; a companion article, Serebriiski et al., describes alternative use of this system in bacteria.