Human renin substrate (angiotensinogen) was purified from outdated bank plasma. Purification procedures included ammonium sulfate precipitation, DEAE-cellulose column chromatography, concanavalin A-Sepharose column chromatography, hydroxylapatite column chromatography, preparative isoelectric focusing and Ultrogel AcA 44 gel filtration. The final recovery was 10% and a specific angiotensin I content of 10.5 .mu.g/mg protein was obtained. Polyacrylamide gel and sodium dodecyl sulfate polyacrylamide gel electrophoresis and analytical ultracentrifugal analyses showed the homogeneity of the purified renin substrate. The MW of 60,900 was determined by sedimentation equilibrium studies. Human renin substrate was a glycoprotein containing 13% carbohydrate. Cystine could not be detected on amino acid analysis. The purified renin substrate showed the isoelectric point heterogeneity (pI, 4.6 and 4.9).