The discovery of a second estrogen receptor (ER), designated ERbeta (NR3A2), has redefined our knowledge about the mechanisms underlying cellular signaling by estrogens and has broad implications for our understanding of regulation of estrogen-responsive tissues. Highly variable and even contrasting effects of estrogens in different tissues seem to be at least partially explained by different estrogen signaling pathways, involving ERalpha (NR3A1) and/or ERbeta. To date, two key conclusions can be drawn from the significant body of work carried out on the specific roles of the two receptor subtypes in diverse estrogen target tissues. First, ERalpha and ERbeta have different biological functions, as indicated by their specific expression patterns and the distinct phenotypes observed in ERalpha and ERbeta knockout (alphaERKO and betaERKO) mice. Second, ERalpha and ERbeta appear to have overlapping but also unique sets of downstream target genes, as judged from a set of microarray experiments. Thus, ERalpha and ERbeta have different transcriptional activities in certain ligand, cell-type, and promoter contexts, which may help to explain some of the major differences in their tissue-specific biological actions. The phenotypes observed for betaERKO mice have suggested certain therapeutic areas to be further explored. The development of ERbeta-selective ligands active in animal disease models indicates new avenues for clinical exploration. ERbeta agonists are being explored and validated as drugs for a growing number of indications. Hopefully, some ERbeta targeted drugs will prove to be efficient in enhancing human health.