We have created four IgG3 mutants without a normal hinge region: (i) m0 without a genetic hinge; (ii) m0/C131S, where Cys-131 in m0 was mutated to Ser; (iii) m0/231C232 (formerly HM-1), where a Cys residue was inserted in m0 between Ala-231 and Pro-232; (iv) m0/C131S/231C232, which is a hybrid of m0/231C232 and m0/C131S. The wild-type IgG3 and all mutants bind 5-iodo-4-hydroxy-3-nitrophenacetyl groups. The wild type and mutants, m15 (with 15 aa in the hinge), m0/231C232, and m0/C131S/231C232, were all positive for complement-mediated lysis, antibody-dependent cellular cytotoxicity mediated by peripheral blood leukocytes, and phagocytosis by U937. m0/C131S/231C232 was only weakly positive and sometimes negative for respiratory burst activity mediated by peripheral blood neutrophils (polymorphonuclear leukocytes), whereas m15, m0/231C232, and wild-type IgG3 were strongly positive. The m0 and m0/C131S mutants were mainly negative for complement-mediated lysis, antibody-dependent cell-mediated cytotoxicity, and phagocytosis by U937 and polymorphonuclear leukocytes. The results indicate that a hinge spacer region is not necessary, but the correct alignment of the two second heavy chain constant regions in the IgG3 molecule by a minimum of one disulfide bond is necessary and sufficient for effector functions.