Microorganisms were among the first tools used for the discovery of biologically active compounds. From recombinant DNA technology emerged important role for microorganisms in pharmaceutical research, the expression of heterologous proteins for therapeutic products or for in vitro high throughput screens (HTSs). Recent developments in cloning, genetics & expression systems have opened up new applications for recombinant microorganisms in screening for nonantibiotic compounds in HTSs. These screens employ microorganisms that depend upon the function of a heterologous protein for survival under defined nutritional conditions. Compounds that specifically target the heterologous protein can be identified by measuring viability of the microorganism under different nutrient selection. Advantages of this approach include a built-in selection for target selectivity, an easily measured end point that can be used for a multitude of different targets, and compatibility with automation required for HTSs. Mechanism-based HTSs using recombinant microorganisms can also address drug targets that are not readily approachable in other HTS formats, including certain enzymes, ion channels & transporters, and protein:: DNA & protein::RNA interactions.