The autolytic cleavage reaction of C3 and C4 and the covalent binding reaction of these proteins, are both aspects of the reactivity of an activated thiolester within these proteins. Autolytic cleavage occurs by internal nucleophilic attack on one face of the planar thiolester, while the covalent binding reaction of the activated proteins follows exposure of the opposite face of the thiolester to attack by external nucleophiles. Although the autolytic cleavage reaction does not occur under physiological conditions, the study of this phenomenon has provided valuable evidence in support of the mechanisms postulated for the physiological covalent binding reactions. The ease with which autolysis can be induced and observed in C3, C4, and alpha 2 M has provided a valuable method for detecting the active forms of these proteins in circumstances where other assays are impracticable, as, for example, in the examination of the uptake of active C3 by lymphocytes. Autolytic cleavage has also been used by Karp and colleagues to produce fragments used in characterizing genetic and biosynthetic variants of mouse C4 and the mouse protein Slp, which is structurally similar to C4. Gross structural comparisons made among C3, C4, and alpha 2 M on the basis of alignment of the autolytic cleavage sites and the protease-activation sites in these proteins were useful in predicting how the alpha-, beta-, and gamma-chains of C4, or the alpha- and beta-chains of C3, were aligned in the single polypeptide chain pro-forms of these proteins. The beta-alpha-gamma alignment deduced for C4 was also found by Goldberger and Colten. Similar alignments of cleavage sites have been used as a basis for evolutionary comparisons of complement proteins and alpha 2 M from species other than man. Although autolytic cleavage has been described only for C3, C4, alpha 2 M, and Slp, it is likely that other proteins will be found that exhibit this phenomenon. A possible candidate is pregnancy-associated plasma protein A (PAPP-A) which resembles alpha 2 M in many respects. The autolytic cleavage reaction will serve as a useful indicator in the detection of other proteins that undergo covalent binding by the mechanism discussed above.