A method, modified from previously reported techniques, for the measurement of 6-hydroxycortisol in human urine is described. 3H-6 β-hydroxycortisol is employed as an internal recovery standard and 1 thin layer chromatogram used for isolation. The accuracy, precision and specificity of the method are documented. Levels are reported in a large group of endocrinologically normal males, females and older children. A positive correlation was observed in the excretion of 6-hydroxycortisol with that of 17-hydroxycorticosteroids and creatinine which supports the use of the ratio, 6-hydroxycortisol/ 17-hydroxycorticosteroids, as an indicator of cortisol metabolism. This ratio was found to be significantly higher in females than males. Ancillary studies support the concept that some of the urinary 6-hydroxycortisol may originate directly from the adrenal glands but the evidence is not consistent and hence this question is unsettled at the present time. © 1968 Holden-Day, Inc.