In an attempt to establish optimal conditions for the hydrolysis of estrogen conjugates in urine with Brown's method, the study was made of the hot acid hydrolysis, enzyme (β-glucuronidase) hydrolysis and solvolysis. The modification of the micro-method according to Salokangas & Bulbrook was reinvestigated. urinary estrogens in fifteen normal male subjects were meseaured with some modified micro-method involving two step hydrolysis (β-glucuronidase hydrolysis and solvolysis). The following results were obtained. 1. Optimal conditions for the hot acid hydrolysis were in 15v/v % HCL concentration at 100℃ for 60minutes incubation. For β-glucuronidase hydrolysis, those were in 2,000units per ml enzyme concentration at 37℃ for 72hours incubation and at 50℃, incubation time was 48hours. Optimal incubation time for the solvolysis was 72hours at 37℃ in 2% H(2)SO(4) concentration with continuous ether extraction. 2. Compared with the hot acid hydrolysis and two step hydrolysis involving β-glucuronidase
hydrolysis and solvolysis, quantities in total estrogens were mostly equal but estradiol fraction in the hot acid hydrolysis diminished little. 3. With the modified micro-method according to Salokangas & Bulbrook, the smallest amounts of pure estrogen could be estimated to 0.015μg and the mean ratios of recovery in this procedure were as follows; estrone 76.7%, estradiol 65.1%, estriol 82.8%. By this method, it was possible to dtermine urinary estrogens untill 0.5μg per day. 4. Urinary estrogens in eleven normal male subjects with 24 to 35years old were as follows; estrone 1.7±0.5μg per day (mean±standard deviation), estradiol 1.7±0.7, estriol 3.5±0.6, total estrogen 7.0±1.0, and in fourth with 60 to 70yers old, estrone 1.3±0.4μg per day, estradiol 2.5±0.4, estriol 2.6±1.1, total estrogen 6.4±1.2.