Complement (C) activation is thought to be critical for the hyperacute rejection of xenografts. We investigated the role of C in the rejection of discordant cardiac xenografts by studying outcome in recipients depleted of C, using a highly purified form of cobra venom factor (CVF) in both a small (guinea pig [GP]-to-rat) and large (pig-to-baboon) animal model. A single dose of 30 or 60 units CVF given i.v. to rats completely abrogated hemolytic C activity for up to 72 hr. The lack of hemolytic C activity correlated with nearly undetectable serum levels of C3. Doses of 30 U/kg daily or 60 U/kg every other day over a 7-day period sustained C depletion without morbidity or mortality. Rats receiving GP cardiac xenografts during CVF therapy had significantly prolonged xenograft survival (88±10 hr in CVF-treated rats vs. 18.6±7.2 min in control rats, P<0.001). Rats that rejected GP xenografts at 4 days posttransplant had higher levels of anti-GP antibodies than control rats, without hemolytic C activity at rejection. This rise in xenoreactive Ig reflected an increase in circulating IgG and IgM against GP antigens recognized before transplantation. Histologic analysis of GP cardiac xenografts taken from CVF-treated rats revealed leukocyte and monocyte margination along blood vessels, beginning at 12 hr posttransplant. Progressive cell infiltration, interstitial hemorrhage, and necrosis were observed over the next 72 hr. Rejected GP xenografts showed diffuse deposition of IgM and fibrin within blood vessels but no evidence of C3 deposition. A nonspecific pattern of IgG deposition was noted. CVF was tested in baboons. Complete C depletion was achieved with a dose of 60 U/kg, and was not associated with any morbidity or mortality. Xenotransplantation of a pig heart was performed in one baboon receiving CVF, 60 U/kg/day, for 2 consecutive days. Xenograft survival was prolonged to 68 hr, compared with 90±30 min in control baboons. Lack of hemolytic activity was noted during engraftment and at rejection. Histology showed evidence of vascular rejection. Immunopathology showed diffuse deposition of IgM, fibrin, and C4, and absence of C3 or membrane attack complex. We conclude that highly purified CVF can achieve marked C depletion with minimal morbidity and no associated fatalities. CVF alone can significantly prolong discordant cardiac xenograft survival. In the GP-to-rat model, the improvement in graft survival achieved with CVF was better than with conventional immunosuppression or isolated acute antibody depletion. The rejection of GP and porcine xenografts under conditions of C depletion and the associated histo/immunopathologic findings suggest that humoral or early cellular effector mechanisms independent of C may be involved in xenograft rejection when the triggering of HAR is prevented.