Proteins from pig brain were screened for high affinity calcium binding by gel filtration of protein fractions previously incubated with calcium-45. Only two proteins from brain exhibited calcium binding in this assay. One of these proteins has been isolated and purified to homogeneity as judged by electrophoresis as a single band in acrylamide gels. The purification procedure employed extraction of a pig brain acetone powder with 0.05 m Tris-HCl, pH 7.4, and fractionation of the proteins in this aqueous extract by ammonium sulfate precipitation, chromatography on ECTEOLA-cellulose, gel filtration on Sephadex G-75, and chromatography on DEAE-cellulose. The molecular weight of the calcium-binding protein was found to be 13,000 as determined by gel filtration. The electrophoretic behavior of this protein was not influenced by calcium or 8 m urea, which do produce electrophoretic heterogeneity of the S-100 protein, which also binds calcium. Amino acid analysis revealed that the protein is rich in glutamic and aspartic acids, threonine, serine, leucine, glycine and lysine. Fractionation of pig liver acetone powder demonstrated that this calcium-binding protein is not present in liver.