A simple and rapid procedure for isolation of the total platelet membrane fraction by chromatography on Sepharose CL 6B has been developed. This method allows a rapid (25-30 min) one-step separation of the membrane fraction on 26 x 150 mm columns with a 20-21 mg recovery. The high degree of purity of membrane preparations was confirmed by a radioligand assay using [3H]adenosine and L-[3H]glutamic acid. The purity of membrane preparations is comparable with that of membrane fractions obtained by standard ultracentrifugation methods. The homogeneity of the membrane fraction was demonstrated by using marker enzymes of plasma, microsomal and mitochondrial membranes. This finding is very important in that it allows the isolation of fractions differing in their protein content with no effect on the method reproducibility. The high utility of the membrane preparations in receptor studies was demonstrated for high affinity binding sites for [3H]adenosine and L-[3H]glutamic acid.