We have previously noted a large difference in the specificity of the covalent binding reaction of human C4-A and C4-B. Here we report data on three other thioester- containing proteins. Human C3 is unreactive with glycine but its reactivity with glycerol (kˊko = 23.0 M–1) is similar to that of human C4-B (kˊko = 15.5 M–1). Human a2-macro- globulin reacts with glycine (kˊko = 206 M–1) in a manner similar to C4-B (kˊko = 119 M–1 ) but its reactivity with glycerol (kˊ/ko = 1.2 M–1) is C4-A like (kˊ/ko = 1.3 M–1). Mouse C4 is C4-B like in its reaction with both glycine (kˊko = 136 M–1) and glycerol (k'/ko = 26.0 M–1). Of these proteins, only C4-A shows a very high rate of reaction with glycine (kˊko = 13,400 M–1). The comparison of the primary structures of these proteins has allowed us to propose the Leu Asp:lie His substitutions at positions 1105 and 1106 in the human pro-C4 molecule as the residues largely responsible for the binding specificities of these proteins. The Leurlle change would not markedly affect the reactivity of these proteins, but may be necessary for allosteric reasons. The Asp in C4-A and His in C4-B seem likely to be the major specificity-defining residues.