Steroidal nuclear receptors (NRs) have been acknowledged as a target binding protein of so-called endocrine disruptors. It is therefore necessary to develop an efficient assay system for screening these endocrine-disrupting chemicals. We here describe the first exemplification of a direct measure of fluorescence intensity for a binding assay of NRs. We designed and synthesized a series of conjugates of 17α-ethinylcarboxyestradiol with carboxyfluorescein, both carboxyl groups of which were cross-linked with α,ω-diaminoalkanes. The resulting fluorescein-linked estradiol derivatives E2(n)cF (n=2, 4, 6, 8, 10 and 12) were evaluated for their fluorescence and receptor-binding characteristics. E2(4)cF and E2(8)cF exhibited the sufficient binding affinity to the recombinant estrogen receptor (ER) in the radiolabel binding assay using [3H]17β-estradiol, and showed excellent fluorescent characteristics in the fluorescence measurements with and without ER. They exhibited sufficiently large specific binding characteristics with adequate Kd- and Bmax-values. When these fluorescent ligands were used as a tracer for the binding assay against the ER, assay data of various compounds were shown to be compatible with those obtained from the ordinary binding assay using [3H]17β-estradiol. The present study clearly shows that measurement of fluorescence intensity, instead of fluorescence polarization, affords an adequate receptor-binding assay system.