In previous studies of monoclonal antibodies (mAbs) against the receptor for epidermal growth factor (EGF) both 528 IgG2a and 225 IgG1 were shown to inhibit growth of A431 cell xenografts in athymic mice. The antitumor activities of the two mAbs were similar and, although they differ in their isotypes, they share many properties. The two mAbs bind to EGF receptors with identical affinities, compete with EGF for binding to EGF receptors, down regulate the receptors identically, block EGF-induced activation of tyrosine protein kinase activity to a comparable degree, and block EGF-induced changes in the proliferation of cultured cells. These similarities in physiological effects permit a direct comparison of the mechanisms of action mAbs of the IgG1 and IgG2a isotypes. We examined in vitro cytotoxicity against A431 cells, using 528 IgG2a and 225 IgG1 mAbs. 528 IgG2a, but not 225 IgG1, demonstrated partial complement-mediated cytotoxicity by the 51Cr release assay and by growth inhibition of cultured A431 cells. 528 IgG2a, but not 225 IgG1, was cytotoxic to A431 cells in the presence of activated peritoneal macrophages, as demonstrated by release of incorporated [3H]thymidine. Neither mAb showed any significant cytotoxicity to A431 cells in the presence of nonadherent spleen cells which contain K-cells. The results of in vitro cytotoxicity experiments suggested that the antitumor activity of 528 IgG2a, but not 225 IgG1, could be mediated by macrophages. This was verified by in vivo experiments in which s.c. tumor cell inocula containing activated macrophages showed enhancement of antitumor effects in animals treated i.p. twice weekly for 3 weeks with suboptimal doses of 528 IgG2a. This enhancement was not observed when 225 IgG1 was used with the same procedure. The results of these experiments suggest that immune mechanisms involving activated macrophages or complement could contribute to the antitumor activity of anti-EGF receptor mAb with the IgG2a isotype, but not with the IgG1 isotype. This observation confirms the findings of others who examined the antitumor activity of IgG2a mAbs in other model systems. IgG1 mAb 225, and possibly IgG2a mAb 528, may prevent growth of human tumor xenografts by altering the physiological functions of the EGF receptor rather than by immune mechanisms.