A novel estrogen receptor (hereinafter referred to as ERβ) was cloned using degenerate PCR primers. A comparison of the amino acid sequence of ERβ with the ‘classical’ ER (ERα) shows a high degree of conservation of the DNA‐binding domain (96%), and of the ligand‐binding domain (58%). In contrast, the A/B domain, the hinge region and the F‐domain are not conserved. Northern blot analysis revealed that ERβ is expressed in human thymus, spleen, ovary and testis. Transient transfections of an ERβ expression construct together with an ERE‐based reporter construct in CHO cells clearly demonstrated transactivation of ERβ by 17β‐estradiol. In addition, the ERα antagonist ICI‐164384 is a potent antagonist for ERβ as well. Interestingly, the level of transactivation by 17β‐estradiol is higher for ERα than for ERβ, which may reflect suboptimal conditions for ERβ at the level of the ligand, responsive element or cellular context.