Summary: By use of Seitz filtration, elution, back filtration, and differential ultracentrifugation techniques, it has been possible to purify β-lysin. The degree of purification when measured as specific bactericidal activity/unit nitrogen was from 1000 to 5000 times that of normal serum. Separation of β-lysin from serum or purified preparations by Millipore filtration was primarily a mechanical process. A comparison of Millipore and Seitz filtration procedures revealed that the latter technique was more efficient for the purification of β-lysin. β-lysin was separated from both agglutinating antibody and complement and was not dependent on either of these serum components for bactericidal activity.