Functionally active guinea pig factor B was purified by a combination of chromatographic steps including Sephadex G-25, QAE A25, QAE A50, CM C50, and Sepharose 4B coupled with purified cobra venom factor. Purified factor B had a m.w. of 106,000 daltons and a single subunit structure. It was heat labile. After cleavage of native B with cobra venom factor coupled to Sepharose 4B in the presence of D̄, the resulting two fragments, the larger one (Bb) and the smaller one (Ba), were further purified. The m.w. of Bb and Ba was determined as 64,000 and 53,000 daltons, respectively, by SDS-PAGE. Neither of the fragments evoked a contraction of guinea pig ileum or histamine release from rat mast cells. Only the smaller fragment Ba (at a concentration of 120 nM) stimulated guinea pig peritoneal polymorphonuclear leukocytes to respond with increased movement. This activity as well as the antigenicity of Ba were heat stable, but were sensitive to trypsin digestion, whereas the antigenicity of Bb was heat labile.