We previously presented a homogeneous noncompetitive assay principle based on quenching of the fluorescence of europium(III) chelate. This assay principle has now been applied to the measurement of 17beta-estradiol (E2) using europium(III) chelate labeled estradiol specific antibody Fab fragment (Eu(III)-Fab) as a donor and E2 conjugated with nonfluorescent QSY21 dye as an acceptor. Fluorescence could be measured only from those Eu(III)-Fab that were bound to E2 of the sample, while the fluorescence of the Eu(III)-Fab that were not occupied by E2 were quenched by E2-QSY21 conjugates. The performance of the assay was tested both in buffer and in the presence of serum. Because approximately half of the Fabs were incapable of binding to E2, the maximum quenching efficiency was only 55%. The lowest limits of detection were 18 and 64 pM in buffer and serum-based calibrators, respectively. The highest measurable concentrations were 0.4 nM in buffer and 1 nM in serum. The presented noncompetitive assay principle requires only one binder, and it can be applied to other haptens as well, providing that a suitable antibody is available.