The previously reported complement-dependent spreading of freshly explanted mouse peritoneal macrophages was further analyzed. Whereas the screening of components of the classical pathway did not reveal any activity, mixtures of human Factors B, D̄ and C3b (after incubation) induced spreading in 50–80% of 5 × 104 glass-attached macrophages within 60–120 min. In order to define the active factor(s) or their fragments 125I-labeled B was added to fresh human serum which was then activated for 60 min, 37° using 5 mg inulin/ml serum. After fractionation of the serum on DEAE- and CM-cellulose and on Sephadex G150 columns spreading activity was found in fractions containing the activated form of B (Bb) and closely followed the distribution of 125I-Bb in the columns' eluates. Fractions containing C3b or Ba lacked activity. Highly purified Bb was prepared from purified B by incubation with D̄, and C3b. Bb was then separated from D̄, Ba and C3b by DEAE-cellulose chromatography.