We developed a new method for measuring an unidentified ketosteroid glucuronide (US-G) detected by the method of Iwata et al. for measuring 17-ketosteroid glucuronides by reversed phase HPLC on a Capcell-Pak C₈ column with three kinds of mobile phase solutions (Iwata method; Clin Chem 35: 795-799, 1989). The Iwata method inadequately separated US-G and two hydroxy 17-ketosteroides, 11β-hydroxyetiocholanolone and 11β-hydroxyandrosterone, and it exhibits insufficient sensitivity for measuring traces of US-G in the urine of healthy subjects. We solved these problems by developing a new method which measures US-G in urine, as a free type by hydrolyzing the glucuronide type enzymatically, by normal phase HPLC on a Capcell-Pak Silica column with one kind of mobile phase solution. By this method, the levels of US excreted as a glucuronide in the urine of healthy subjects and of patients with Cushing's syndrome were determined as proportions of the levels of 11β-hydroxyandrosterone. The average daily urinary excretion of US was 971μg (125-4, 995μg) in patients with Cushing's syndrome (n=22: two males and 20 females aged 26 to 65 years), and 34μg (0-141μg) in healthy subjects (n=63: 49 males, and 14 females aged 21 to 54 years), and the differences were clearly significant. However, there were no differences between the urinary US levels of patients with pituitary adenoma and patients with adrenal adenoma. Furthermore, no US was detected in the urine of patients with aldosteronism (two males and eight females aged 34 to 61 years). The daily level of urinary US excretion in two of the patients with polycystic ovary syndrome was 159 and 142μg, but no US was detected in the other two patients.