This article describes the isolation and characterization of a previously unknown, leucine-rich 3.1 S-alpha2-glycoprotein from human serum. The starting material was Supernatant II, which is a byproduct in the large-scale preparation of albumin and gamma-globulin by the ethacridine lactate/ammonium sulfate procedure. The purified protein is homogenous both in carrier-free and molecular-sieve electrophoresis. Its electrophoretic mobility indicates that it belongs to the alpha2-globulins. Isoelectric focussing splits it into 4 bands with isoelectric points between 3.8 and 4.1. In the ultracentrifuge it sediments in a single band at 3.1 S. The molecular weight determined by equilibrium sedimentation is 49 600+/-4 000. Subunits were not detected. Chemical analysis reveals it to be a glycoprotein with a carbohydrate content of 23%. The amino acid content is unusual in that the leucine content is almost 17%, ie about every fifth amino acid is a leucine. The average concentration of the leucine-rich 3.1 S-alpha2-glycoprotein in human serum was determined by a quantitative immunological method as 2.1 mg per 100 ml. The protein is not related to any of the previously known well characterized serum proteins.