Upon activation of the complement system in guinea pig serum by endotoxin, a factor chemotactic for polymorphonuclear leukocytes is generated. This chemotactic factor has an approximate molecular weight (M.W.) of 15,000 and is independent of three minor chemotactic factors (M.W. τ;150,000, ∼68,000 and <4000) present in normal, heat-inactivated or EDTA-treated guinea pig serum. Purified, radiolabeled guinea pig C′3 and C′5 were used to determine whether the 15,000 M.W. chemotactic factor generated in serum was derived from either of these components. Endotoxin was incubated with guinea pig serum to which had been added either 125I C′3 or 125I C′5. Gel filtration elution profiles of the reaction mixtures revealed that both C′3 and C′5 were cleaved in endotoxin-treated serum. Moreover, the 15,000 M.W. chemotactic factor was identified with a cleavage product of C′5 but not of C′3. In addition, rabbit anti-guinea pig C′5, but not anti-C′3, significantly inhibited the activity of the 15,000 M. W. chemotactic factor. We conclude that the majority of chemotactic activity found in whole guinea pig serum after interaction with endotoxin is a product cleaved from C′5.