We describe a highly efficient and sensitive yeast-based screening method for isolating human estrogen receptor alpha (ERalpha) mutants with altered trans-activation activity. This method takes advantage of the fact that estrogen receptor is a ligand-activated transcription factor, and links the transactivation activity of estrogen receptor to the growth rate of yeast cells. We used this method to screen a library of human ERalpha mutants created by random mutagenesis of the ligand binding domain of human ERalpha in the presence of ligand 17beta-estradiol (E(2)). We isolated several human ERalpha mutants with significantly altered trans-activation activity toward E(2) in yeast cells. We also used this method to screen a library of chemical compounds and showed that it can be used to rapidly identify estrogenic compounds and the different cell growth rates for these estrogenic compounds correlated well with their relative binding affinities. Thus, this method is suitable for selecting novel estrogenic compounds and estrogen receptor mutants. In principle, this method might also be used to isolate mutants of any nuclear receptors with altered trans-activation activity, which may greatly facilitate their structural and functional studies.