High sensitivity radioimmunoassay of 3beta,7beta-dihydroxy-5-androsten-17-one (7beta-OH-DHEA) has been developed and evaluated. The method is based on polyclonal rabbit antisera raised against its 19-O-(carboxymethyl)oxime bovine serum albumin conjugate and bridge- and position homologous [125I]iodotyrosine methyl ester as a tracer. Alternatively, [3H]tracer has been prepared, which was recognized by the antiserum as well, but the assay sensitivity was lower. The identity of measured immunoreactive material was confirmed by high performance liquid chromatography which separated 7beta-OH-DHEA from its 7alpha-isomer. Using radioiodinated tracer, the sensitivity of the method was 3.48 fmol (1.06 pg) per tube, the mean recovery of standard added to steroid-free serum was 98.5%. Free (unconjugated and not-esterified) 7beta-OH-DHEA amounted in average 5.8% of the total 7beta-OH-DHEA present in human serum. It was measured in 42 normal subjects (28 females and 14 males) and in 92 randomly selected patients with various endocrinopathies. The mean values +/- SD in normals were 2.05 +/- 1.02 nmol l(-1), the broad range of values from undetectable levels to 10.3 nmol l(-1) was found in the patients. Serum levels of free 7beta-OH-DHEA in the patients significantly correlated with DHEA and its sulfate.