Activated Cls, the enzymatically active subunit derived from the first component of human complement, has been shown to cleave the fourth component of human component (C4) into relatively heavy (C4b) and light (C4a) fragments as ascertained by sucrose density gradient ultracentrifugation and gel filtration. These fragments remain associated at neutral pH but dissociate upon acidification to pH 3.5. The estimated molecular weight of C4a is 8600. Cleavage of C4 by Cls̄ was not influenced by the further presence of C2 or C2 and C3 in reaction mixtures. Isolated C4a and C4b could not be reassociated at neutral pH. C4a failed to contract preparations of guinea pig ileum or rat uterus and did not mediate a wheal and flare response in human skin under the conditions tested.