Fluorescence polarization immunoassay (FPIA) offers a good alternative to isotopic methods for the determination of drug and hormone levels in biological fluids. We applied it to estradiol, using a fluoresceinated derivative of estradiol and the IgG fraction of a highly specific rabbit antiserum. After determining the optimal operational conditions, FPIA was used for measuring urinary concentrations of free estradiol in normal and pregnant women, after a purification step on a reversed phase cartridge. The method turned out to be rapid (1 min to reach the equilibrium state) and accurate.