A method for the extraction and quantification by high performance liquid chromatography (HPLC) of non-labelled benzodiazepines partitioned into biological membranes has been developed. The benzodiazepine (BZD) was partitioned in a synaptosomal membrane-buffer system. The membranous pellet was separated by centrifugation and from this pellet, previously submitted or not to a proteolytic treatment and resuspended in the same buffer, the BZD was extracted by the addition of the proper volume of ethyl acetate, i.e. that capable of extracting at least 99.5% of the total drug in the aqueous phase. Optimal conditions for maximum proteolysis were: incubation of membranes at a final concentration of 1 mg protein/mL in the presence of 0.08 mg of Protease type I per mL for 90 min. at 45 degrees C. Although the efficiency of the proteolysis was demonstrated not only by kinetic but also by electrophoretic evidence, recovery of BZD in the organic solvent was not increased by the enzymatic treatment at a mass BZD/protein ratio in the range 4-4.6 10(-5) micrograms BZD/mg protein. For the HPLC quantification a reversed phase ODS column was used with methanol:water (1:1) plus 3.5% acetic acid as the mobile phase at a constant pressure and 1 mL/min flow rate. The UV detector was calibrated at 265 nm. Calibration curves were constructed by plotting peak height or peak area vs. nmoles BZD and adjusted to straight lines by a regression analysis by the least squares method. The peak height was selected as the detection method. Intra- and inter-assay precision did not exceed 10% and the total recovery was 100%.