In-vitro studies of chemotaxis of mononuclear leukocytes (MNLs) were performed to characterize humoral mediators governing the accumulation of these cells at inflammatory sites. Two humoral factors that attract MNLs in vitro were found. The first was a heat-labile factor (mol wt, 90,000) present in untreated guinea-pig serum and plasma. The second factor was heat-stable, of low mol wt (13,000–15,000) and was found in guinea-pig serum that had been treated with immune complexes, endotoxin, or cobra-venom factor. Inhibition or inactivation of the complement (C′) system prevented generation of this latter MNL chemotactic activity. In addition, the data showed that the MNL chemotactic factor of low mol wt, generated in vitro in guinea-pig serum, was specifically inhibited by rabbit antiserum to guinea-pig C′5 and C′5a. Thus, cleavage of C′5 by activation of the C′ system in whole serum releases C′5a, the biologic activities of Which include increasing vascular permeability, contraction of smooth muscle, chemotaxis of polymorphonuclear leukocytes, and, as shown by these data, chemotaxis of MNLs.