The optimum conditions for hydrolysing conjugated metabolites of steroid hormones in bovine urine were performed with Helix pomatia juice, b-glucuronidase from bovine liver and preparations of limpets and abalone entrails using response surface methodology. The experimental design and empirical modelling used allowed us to assess the main effects of factors (time, temperature, pH and enzyme quantity) and to predict the optimum conditions for each enzyme preparation. Confirmatory experiments were applied to check the predicted values and to validate the model. The comparison of the enzyme preparation efficiency for various conjugate steroids and the study of possible by-product synthesis led us to select abalone entrails to hydrolyse natural dehydroepiandrosterone, etiocholanolone, epitestosterone; 17a-estradiol and estrone in bovine urine. The optimum conditions were found to be 20 h at 42 °C with the pH adjusted to 5.2 and using 12 000 units of enzyme preparation.