Abstract Fluorescent derivatives of estradiol-17β (E2), desoxycorticosterone (DOC) and prednisolone (Pr) were synthesized by coupling E2-17β-hemisuccinate (E2-HS), DOC-21-HS and Pr-21-HS respectively to N-fluoreseeinyl-5,N'-(6-amino)-hexyl-thiourea. The long chain of C and N atoms between the steroid and fluorescein was introduced to avoid steric hindrance of the steroid-receptor interaction. The binding affinity of E2-fluorescein-conjugate (E2-F1) was determined by competitive inhibition of the specific [ 3H]-E2 binding to rabbit uterine cytosol receptors. Dissociation constants were found to be 0.8 nM for E2 and 1.5 nM for E2-F1. In the same way, affinity of the DOC-fluorescein-conjugate (DOC-'F1) binding to the progesterone receptor of rabbit uterus was measured using [ 3H]-R 5020 as radioactive ligand. K D was 9.7 mM for DOC-F1 compared to 2.3 nM for progesterone. Pr-fluorescein-conjugate (Pr-F1) bound to the glucocorticoid receptor with a K D of 7.3 nM compared to 3.4 nM for dexamethasone.