A rapid and very sensitive enzymatic assay of estrogens was developed using the transhydrogenase reaction of the human estradiol-17 beta dehydrogenase and bioluminescence for the quantification of NADH accumulated during the first reaction. The assay requires two steps: first, the addition of transhydrogenase buffer, and a few minutes later the addition of the bioluminescence reagent. The method can determine 0.1 to 50 pg of estrogens in the assay, in 1 h, with a coefficient of variation of 5%.