A heterologous immunoassay for 2-hydroxyoestrogens1 has been established in which antibodies raised against 2-hydroxyoestradiol-17-succinyl-BSA serve as binding protein and 2-hydroxyoestrone-17-cmo\x=req-\[125I]iodohistamine as radioligand. Lipophilic serum components competing for binding sites in this system were defined as 'total 2-hydroxyoestrogens'. The underlying assumption of specificity was supported by the pattern of cross-reactivity evaluated with structural related steroids and o-diphenols and by the fact, that an additional chromatography of the serum extracts preceding the competing reaction had little if any effect. Sensitivity: 2.8 \m=+-\1 pg/tube; accuracy: Y = 0.91x + 2.2; r = 0.989; precision: 5.8% intra-assay; 6.5% inter-assay. The following concentrations ( \ m=+-\ standard deviation) were found in the sera of healthy subjects.Young men: 29 \m=+-\5 pg/ml (n = 11); women follicular phase: 32 \m=+-\8 pg/ml (n = 25); luteal phase: 53 \m=+-\13 pg/ml (n = 23); postmenopausal women: 13 \m=+-\4 pg/ml (n = 10); pregnant women 11th\p=n-\20th week: 70 \m=+-\16 mg/ml (n = 64); 36th\p=n-\40thweek: 240 \m=+-\23 pg/ml (n = 40); newborn cord blood: 604 \m=+-\43 pg/ml (n = 48).2-Hydroxyoestrogens have been recognized as major metabolites of oestrone and oestradiol in both laboratory animals and humans. The opinions on their physiological significance are still at vari¬ ance (Ball & Knuppen 1980). The assay of cate¬ choloestrogens in biological fluids and tissue by the classical methods of steroid separation and detec¬ tion is hampered by their chemical instability. They are highly susceptible to attack by oxidants, especi¬ ally in alcaline media. In addition, the classical methods are time consuming, expensive and not applicable for routing determinations. These pro¬ blems have been mastered by the development of suitable radioimmunoassays (Chattoraj et al. 1978;Ball et al. 1978). Methods currently available utilise tritium labelled radioligands, which are not avail¬ able commercially. Their syntheses are lengthy and tedious with generally small yields. In establishing a sensitive and specific radioimmunoassay of serum catecholestrogens we preferred the use of 125I-labelled radioligands, which offer the advan¬ tages of higher specific activities and cost-saving counting for at least 3 months after preparation. In order to obtain a very high sensitivity of the assay we utilized a heterologous bridge system employing anti-2-hydroxyoestradiol-17ß-succinyl-BSA-serum and 2-hydroxyoestrone-17-cmo-[125I]-iodohistamine. Preliminary results have been pub¬ lished previously (Berg et al. 1981).1 The following trivial names and abbreviations were used: 2-hydroxyoestrone = 2,3-dihydroxy-l,3,5(10)-oestratrien-17-one, 2-hydroxyoestradiol = l,3,5(10)-oestratriene-2,3,17ß-triol, 2-hydroxyoestriol = l,3,5(10)-oestratriene-2,3,16a,17ß-tetrol, oestradiol-17ß = 1,3,5(10)-oestratriene-3,17ß-diol, oestrone = 3-hydroxy-1,3,5(10)-oestratrien-17-one, oestriol = l,3,5(10)-oestratriene-3-16a, 17ß-triol, 2-methoxyoestrone = 2-methoxy-3-h...