The human estrogen receptor ligand-binding domain (hER-E/F), including the distal F domain, has been expressed to high levels in a soluble, active form in Escherichia coli in order to facilitate biophysical studies. The ability of a series of vectors incorporating strong transcriptional and translational signals to provide an efficient expression system for hER-E/F was investigated. High-level expression was obtained from all of the vectors used in the study. Although the majority of hER-E/F protein was produced in insoluble form under standard bacterial culture conditions, hER-E/F could be produced in soluble, biologically active form by altering the sequence of the expressed protein and by varying the host culture conditions. Several parameters, including the presence of a His tag, growth temperature, and addition of ethanol and 17 beta -estradiol to the growth medium were shown to have a positive effect on production of soluble hER-E/F. An optimized expression system capable of producing from 25 to 35 mg of biologically active hER-E/F in 1 liter of cell culture was designed, and a simple, rapid purification protocol for hER-E/F produced in this system was developed. Characterization of purified hER-E/F by Edman degradation and mass spectrometry verified the identity of the protein. The K-D for 17 beta -estradiol binding to purified hER-E/F was determined to be 0.6 +/- 0.1 nM. The parameters controlling soluble, heterologous protein production observed in this study may be generally applicable to the expression of other heterologous proteins in E. coli.