We have established highly sensitive and specific androgen and estrogen reporter cell lines which we have named AR (androgen receptor) and ERα (estrogen receptor alpha) CALUX® (Chemically Activated LUciferase eXpression), respectively. Both bioassays are member of a panel of CALUX reporter cell lines derived from the human U2-OS osteosarcoma cell line, all using highly selective reporter constructs based with a basal promoter element linked to multimerized. response elements, allowing efficient and specific measurement of compounds interfering with androgen, estrogen, progesterone, and glucocorticoid receptors. The AR CALUX bioassay contains the human androgen receptor and a luciferase reporter construct containing three androgen-responsive elements coupled to a minimal TATA promoter. This cell line was characterized by its stable expression of AR protein, its highly selective response to low levels of different natural and synthetic androgens, and its insignificant response to other nuclear hormone receptor ligands such as estrogens, progestins, and glucocorticoids. The EC50 of dihydrotestosterone (DHT) was found to be 0.13 nM, consistent with the high affinity of this ligand to the human AR. Flutamide, cyproterone acetate, and the environmental contaminants vinclozolin, DDT, methoxychlor, its metabolite HPTE, and penta-BFR showed clear antagonistic activity in the AR CALUX bioassay, competitively inhibiting DHT-mediated transactivation. The established AR CALUX bioassay proved to excel in terms of easy cell line maintenance, high fold induction range (typical 30 times over solvent control), low minimal detection limit (3.6 pM), and high androgen selectivity. Potential applications such as testing the androgenic or estrogenic activity of pure chemicals and pharmaceuticals and complex mixtures (environmental, food, feed, and clinical) are discussed. © Society of Toxicology 2005; all rights reserved.