Abstract A method for the simultaneous determination of eight major corticosteroid hormones and precursors in 0.5–2.0 ml of plasma has been developed and is described in detail. After extraction of the unconjugated steroids from plasma to which tritiated steroid tracers had been added, progesterone (P), 11-deoxycorticosterone (DOC), 17-hydroxyprogesterone (17-OH-P), corticosterone (B), 11-deoxycortisol ( S), aldosterone (A), cortisone (E) and cortisol (F) are simultaneously separated using eight mechanized 60 cm Sephadex LH-20 columns in parallel. Each of the isolated steroids is quantitated by radioimmunoassay after saving an aliquot for recovery counting. The combination of Chromatographic separation and appropriate antiserum resulted in high assay specificity. Water or steroid-free plasma blanks were undetectable, with sensitivities of the assays ranging between 0.020 (A) and 0.370 (E) ng/ml. There was very satisfactory precision and accuracy, with inter-assay coefficients of variation below 16.5% and linear relationships between added and found, respectively. Normal values are reported for males, females (follicular phase) and a small group of prepubertal children, revealing sex differences between the adults studied with higher A, B and P levels in females and higher 17-OH-P and E levels in males. The mineralocorticoids A and DOC were considerably higher in children than in adults. The highly practicable method is particularly useful in pediatric and experimental endocrinology.