By the year 2005, the Human Genome Project is expected to have mapped and sequenced all of the estimated 100,000 genes that encode the various proteins found in human cells. Defining the role of each gene, and using that information to redirect its action when therapeutic intervention is required, is one of the major assignments for molecular medicine and molecular urology in the post-genome era. This challenge to determine gene function, and to do it cost-effectively and on a large scale, has driven development of new technologies that can more efficiently flag genes that are likely targets for therapeutic intervention. Yeast-based genetic assays that detect protein-protein interactions in vivo offer many of the features required of a practical "gene-flagging" strategy for identifying genes that might be functionally manipulated to achieve therapeutic goals. In the past few years, the yeast-based assays collectively referred to as "two-hybrid interaction traps" or simply "two-hybrid systems" have become increasingly important tools for experimental analysis of gene function. This review presents an overview of the principles of yeast-based two-hybrid analysis, examines some specific applications of the technique of interest to urologic investigators, and discusses some key points that a basic urologic investigator new to the technology would want to consider when designing or evaluating a yeast-based two-hybrid project.