This study describes the enzymatic hydrolysis of urinary conjugates of cortisol, cortisone, tetrahydrocortisol, allotetrahydrocortisol, and tetrahydrocortisone with beta-glucuronidase preparations from Helix pomatia and Ampullaria. The objective of the present studies was to find optimal hydrolysis conditions for these conjugated steroids. Assay of the isolated steroids was carried out by GC-MS using deuterium-labeled compounds as internal standards. The allotetrahydrocortisol conjugate was clearly the hardest to hydrolyze with enzyme from Helix pomatia and required increased enzyme concentration and prolonged incubation. Hydrolysis of a urine sample for 2.0 h with the simultaneous use of 3400 units/ml Ampullaria and 5400 units/ml Helix pomatia enzymes in 0.5 M acetate buffer at 55 degrees C achieved more complete cleavage of the urinary conjugates of the five steroids examined. It is thus advantageous to use the Ampullaria and Helix pomatia enzymes in combination to obtain the highest yield in the urinary corticosteroid assay.