Human estrogenic 17beta-hydroxysteroid dehydrogenase (17beta-HSD type 1) catalyzes the final step in the synthesis of active estrogens that stimulate the proliferation of breast cancer cells. Based on the initial premise to make use of the binding energies of both the substrate and cofactor sites, and molecular modeling starting from the enzyme structure, several estradiol-adenosine hybrids were designed and synthesized. Among these hybrids, EM-1745 with a linker of 8-CH2 groups is proved to be the best competitive inhibitor with a Ki of 3.0 +/- 0.8 nM. The crystal structure of the EM-1745 enzyme complex at 1.6 A provides evidence at atomic resolution of strong interactions between both the steroid and cofactor moieties and the enzyme molecule, as illustrated by a deltaA-weighted 2Fo-Fc electron density map contoured at 3.0 delta. The substrate entry loop is further stabilized in this complex compared with previous complexes of the enzyme. These results confirm our initial strategy of combining studies of structural biology and enzyme mechanism in the inhibitor design, which may be applied to other steroidogenic enzymes involved in human diseases.