A radioimmunoassay for daidzein was established, based on polyclonal antibodies against daidzein-4'-O-(carboxymethyl)ether-BSA. The sensitivity of the assay was 0.4 pg/tube; the intra- and interassay coefficients of variation varied from 4.1 to 11.5% and from 5.6 to 21.7%, respectively, depending upon the method (direct or extraction) and concentration of daidzein in the sample. The cross reactivities with other chemically related compounds, with the exception of 4'-derivatives of daidzein, were 2.4% for dihydrodaidzein, 1.3% for genistein, 1.5% for biochanin A, and 1.6% for equol, respectively. The method was used for measurement of daidzein levels in 105 normal human subjects and in three volunteers after consumption of a meal prepared from 125 g of cooked whole soybeans. The daidzein values obtained following diethyl ether extraction of human sera was only 8% of that obtained by direct radioimmunoassay. We suggest that this difference is caused by cross-reacting daidzein 4'-glucuronides and -sulfates present in serum. Using ether extraction, the basal serum levels of free daidzein were 0.11 ng/mL (0.43 nmol/L), with 14 subjects showing no detectable levels. Levels were detectable in all subjects with the direct assay with a mean value of 7.1 ng/mL (28.0 nmol/L). Peak levels were reached 4 hours after ingestion of the soybeans. The levels were 10.3 +/- 3.6 ng/mL (40.4 +/- 14.3 nmol/L) for free daidzein and 129.4 +/- 36.1 ng/mL (509 +/- 142 nmol/L) for total immunoreactive compounds. After 24 hours, the levels were still clearly distinguishable from the basal levels; the concentrations were 0.43 +/- 0.15 ng/mL (1.69 +/- 0.59 nmol/L) and 24.36 +/- 6.07 ng/mL (95.9 +/- 23.9) for free and total immunoreactive material, respectively. It is concluded that this is the first immunoassay for a phytoestrogen in human biological fluids, and for the first time serial assays of unconjugated daidzein in plasma have been possible.