Golgi-enriched microsomal membrane fraction was prepared from bovine liver. Sodium choleate extract of this membrane preparation was subjected to fractionation using Sepharose gel covalently bonded with tyrosine-O-sulfate. SDS gel electrophoresis of the fractionated sample revealed the presence of a major protein with an apparent molecular weight of 175,000. The protein appears to be specific for tyrosine-O-sulfate as it binds neither the unmodified tyrosine nor the structurally similar tyrosine-O-phosphate. pH-dependence study showed the binding of the protein to tyrosine-O-sulfate-Sepharose gel to be strong from pH 8.0 down through 6.0. At pH 5.5, the binding affinity became dramatically reduced. A similar tyrosine-O-sulfate-binding protein was also detected in the choleate extracts of the Golgi-enriched microsomal membrane fractions prepared from bovine pancreas and from both liver and pancreas of dog.