A bacterial complementation assay has been developed for the rapid screening of a large number of compounds to identify those that inhibit an enzyme target for structure-based inhibitor design. The target enzyme is the hypoxanthine phosphoribosyltransferase (HPRT). This enzyme has been proposed as a potential target for inhibitors that may be developed into drugs for the treatment of diseases caused by several parasites. The screening assay utilizes genetically deficient bacteria complemented by active, recombinant enzyme grown in selective medium in microtiter plates. By comparing absorbance measurements of bacteria grown in the presence and absence of test compounds, the effect of the compounds on bacterial growth can be rapidly assayed. IC50 values for inhibition of bacterial growth are a reflection of the ability of the compounds to bind and/or inhibit the recombinant enzyme. We have tested this bacterial complementation screening assay using recombinant HPRT from the parasites Plasmodium falciparum and Trypanosoma cruzi, as well as the human enzyme. The results of these studies demonstrate that a screening assay using bacterial complement selection can be used to identify compounds that target enzymes and can become an important part of structure-based drug design efforts.