17beta-Hydroxysteroid dehydrogenases (17beta-HSDs) are substrate- and tissue-specific isoenzymes that regulate activation and inactivation of steroid hormones. Up-regulation and downregulation in expression of 17beta-HSDs are linked to onset of many steroid-dependent diseases, such as colon, prostate, and breast cancer; thus 17beta-HSDs are potential drug screening targets. Currently their enzymatic activities are usually measured using laborious chromatographic separations followed by radioactive detection of substrate and product. We have previously reported the use of a homogeneous luminescence resonance energy transfer-based immunoassay for 17beta-estradiol in screening of potential inhibitors of 17beta-HSD type 1 (17beta-HSD-1). By replacing the previously used cell-based enzyme reactions with recombinant enzyme reactions the sensitivity of the screening assay improved considerably. In addition, the single assay was able to detect the influence of a tested compound not only on 17beta-HSD-1 but also on 17beta-HSD type 2 (17beta-HSD-2), catalyzing the opposite reaction. The screening results of the tested molecules obtained from the optimized immunoassay were very similar when compared with the results of high performance liquid chromatography separation analysis. The Z factors were 0.79 and 0.83 for 17beta-HSD-1 and 17beta-HSD-2 assays, respectively. Thus the immunoassay measuring samples converted with the recombinant enzymes was a very suitable method for primary high throughput screening, and it could be used also in further characterization of potential drugs.