Human platelet glycoprotein IX is a small (Mr 17,000) surface glycoprotein present in the plasma membrane as a 1:1 non-covalent complex with glycoprotein Ib (Mr 165,000). Glycoprotein IX was purified to near homogeneity by sequential wheat germ agglutinin and immuno-affinity chromatography, followed by gel filtration chromatography under denaturing conditions. Purified glycoprotein IX was characterized by determination of its amino acid composition and its NH2-terminal amino acid sequence (thr-lys-asp-xxx-pro-ser-xxx-leu-thr-(thr)2-arg-ala-(leu)-(glu)-xxx-met- gly), and monospecific anti-glycoprotein IX antibody was prepared. The study outlines a useful approach for separating and characterizing glycoprotein IX, free from other members of the glycoprotein Ib complex.