The transactivational property of natural and synthetic chemicals via medaka vitamin D receptor β subtype (VDRβ) was investigated after the development of a stable cell line expressing a Gal4-VDRβ fusion protein for reporter gene assay. Members of vitamin D class, including 1α, 25- dihydroxyvitamin D₃ (1,25VD3) were specifically detected as agonists in our system. Although other steroids and chemicals used in the present estimation induced no agonistic response, 10 compounds displayed antagonistic or synergistic activity. Spironolactone, which is an antagonist of corticoid receptors in mammals, competitively inhibited the transactivity of 1,25VD3 by over 80% in a dosedependent manner. Mifepristone and cyproterone acetate were also detected as antagonists, but they significantly acted only at 10µ. Pregnenolone and raloxifene dose-dependently enhanced the activity of 1,25VD3 at EC₅₀ to the maximum level. Diethylstilbestrol, 17α-ethynylestradiol, genistein, and stanozolol were also synergists, but their potency was low. Interestingly, dibutyltin dichloride, which is used as a stabilizer in the production of polyvinyl chloride plastics, produced greater response than maximum effect of 1,25VD3 although the concentration-response curve was not typically sigmoidal. In the present study, we successfully developed a stable reporter gene assay, which allows assessment of the vitamin D-like chemicals toward the medaka VDRβ.