In this report we describe the construction of anti-5-iodo-4-hydroxy-3-nitrophenacetyl (NIP) mouse/human immunoglobulin (Ig) G4 chimeric molecules with altered amino acid residues in the CH2 domain. Three mutants are described. Gln-268 is substituted by His in gamma 4 Q268H, Ser-331 is substituted by Pro in gamma 4 S331P, and in gamma 4 Q268H/S331P both residues are substituted. The ability of the mutant molecules to induce complement-mediated cell lysis (CML) and phagocytosis by Fc gamma RII- and Fc gamma RIII-bearing polymorphonuclear leukocytes (PMN) were measured. In CML, gamma 4 Q268H was inactive, but both gamma 4 S331P and gamma 4 Q268H/S331P were active provided that the antigenic density on the target cells was high. In phagocytosis mediated by PMN, the mutants gamma 4 S331P and gamma 4 Q268H/S331P were both active only when complement was introduced. gamma 4 Q268H was not active in phagocytosis under any conditions. We conclude that His-268 in human IgG molecules does not modulate CML activity or phagocytosis mediated by Fc gamma RII and/or Fc gamma RIII. Pro-331 rescues CML activity in IgG4 molecules when the epitope density on the target cells is high, but does not affect Fc gamma RII/Fc gamma RIII-mediated phagocytosis. In this manner the mutants gamma 4 S331P and gamma 4 Q268H/S331P mimic human IgG2. This could indicate a structural similarity between IgG2 and these mutant molecules that distinguish them from both IgG1 and IgG3.