Fluorescence resonance energy transfer has been studied between lanthanide(III) chelates as donors and protein-coupled CdTe semiconductor nanoparticles as acceptors. Wide excitation spectra and large Stokes shift of semiconductor nanoparticles and timeresolved fluorescence detection were shown to provide a combination for successful energy transfer assay. Different intrinsically fluorescent europium(III) and terbium(III) chelates coupled to single biotin molecules were studied for optimal energy transfer with streptavidin labeled semiconductor nanoparticles. No significant differences between the studied chelates were observed. The strength of the methodology was demonstrated in a clinically relevant competitive and separation-free immunoassay of estradiol, where subnanomolar limit of detection was achieved with the coefficient of variation 2-11%. The data suggested that relatively short distance was needed to obtain adequate energy transfer. Therefore, biomolecules were coupled onto the semiconductor nanoparticles without any spacers.