To facilitate analysis of protein-protein interaction interfaces, we devised a novel yeast genetic screening method, named the “one plus two-hybrid system”, for the efficient selection of missense mutations that specifically disrupt known protein-protein interactions. This system modifies the standard yeast two-hybrid system to allow the operation of dual reporter systems within the same cell. The one-hybrid system is first used to select the intact interacting partner (prey), resulting in the positive selection of informative missense mutants from a large library of randomly generated mutant alleles. Then, in a second screening step, interaction-defective prey mutants for a given protein are selected using the two-hybrid reporter system among the isolated missense mutants. We used this method to characterize the interactions between unliganded nuclear receptors (NRs) and the conserved motif within the bipartite NR-interaction domains (IDs) of the NR corepressor (N-CoR), and identified the specific residues of N-CoR-IDs required either generally for optimal NR binding or to interact with particular NR. This efficient and rapid method should allow us to quickly analyze a large number of interaction interfaces.