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A new method of assay was applied to the determination of pregnanediol in urines from patients in whom the presence of a placenta or a corpus luteum could be excluded. The normal variation of pregnanediol content in such urines was examined. It was shown that operative stress or the intravenous administration of corticotrophin caused an increased output of pregnanediol. Quantitatively significant amounts of urinary pregnanediol could not be detected after adrenalectomy nor did adrenalectomized patients respond to corticotrophin by an increased pregnanediol output. It is considered that the adrenal contributes to the pregnanediol found in urine and that the determination of urinary pregnanediol may be of value in the study of adrenal function.