Pretreatment of fenugreek (Trigonella foenum-graecum L, Fabaceae) seeds was achieved using the step of defatting and acid hydrolysis. The obtained hydro-isolate (HI) was used as initial feed for diosgenin isolation by supercritical CO2 extraction (SC-CO2). Preliminary tests performed at conditions resulting in SC-CO2 density ranging from 800 to 920 kg/m(3) indicated that at some pressure, temperature as well as consumption of supercritical fluids the optimal working conditions for diosgenin isolation could be determined. For this purpose, the following range of working conditions of SC-CO2 were tested by using Central Composite Rotatable Design (CCRD) and Response Surface Methodology (RSM): pressure from 16.6 to 33.4 MPa, temperature from 30 to 50 degrees C and consumption of SC-CO2 from 11.6 to 28.4 g(co2)/g(dm). The result of this investigation indicated that maximum yield of 0.81 mg diosgenin from 1 g of fenugreek seeds on dry basis (about 1.3% of total extract) at 24.6 MPa, 43.5 degrees C, and 20.87 g(co2)/g(dm) could be obtained. (C) 2015 Elsevier B.V. All rights reserved.