The ingestion of Solanum glaucophyllum (SG) causes a calcinosis of cattle named Enteque Seco (ES). The toxic principle is the 1,25-(OH)(2)D(3), mainly conjugated as glycoside. This study aims to validate a simple novel method of evaluation of the VDA of SG leaves. Aqueous extracts of SG were purified using C(18) minicolumns and assayed by RIA with an antibody raised in rabbits by injection of the acid-C22, 1alpha-(OH)Vitamin D(3). Data were expressed as glycoside equivalent to 1,25-(OH)(2)D(3) in ng/g of dry leaves. We compared this data with 1,25-(OH)(2)D(3) levels measured, in the same samples, by liquid chromatography (HPLC) after enzyme cleavage. This procedure involved the incubation of SG leaves with rumen fluid, followed by C(18)-OH solid phase extraction. The 1,25-(OH)(2)D(3) fraction was run by HPLC and detection was achieved using a photodiode array detector. Data were expressed as micrograms of 1,25-(OH)(2)D(3)/g dry leaves. A significant regression of 1,25-(OH)(2)D(3) levels (Y) as a function of glycoside RIA 1,25-(OH)(2)D(3) equivalents (X) was found: Y=12.02+0.35X [R=0.81; P=0,0002; N=15], allowing us to conclude that this novel assay could be used to estimate the amount of this active principle contained in SG leaves.