Abstract A method is described for the measurement of aldosterone in urine and plasma of rats and the sensitivity, specificity, accuracy and precision of the assay are assessed. The method includes one extraction step by methylene chloride, purification of the extract by one thin layer chromatography and determination of the purified aldosterone by radioimmunoassay. [ 3H]-Aldosterone is used for the radioimmunoassay as well as for the determination of the recovery of the individual sample. A modified calculation of the radioimmunoassay results is described to correct for the varying amounts of [ 3H]-aldosterone of the recovery preexisting in the unknowns but not in the standards of the calibration curve. Normal values of plasma aldosterone concentration in adult male Sprague-Dawley rats (23–288 pg/ml depending on the time of day) as measured with our assay are in good agreement with the values reported in the literature. Normal values of urinary aldosterone excretion rate in these rats (3.9–5.8 ng/24 h) are much lower than those reported in the literature possibly due to the higher specificity of the present assay.