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As pigs homozygous for α1,3-galactosyltransferase gene-knockout (GT-KO) are available, primate antibodies to pig non-Gal antigens can be studied.Sera from 56 baboons were tested for binding of IgM and IgG to peripheral blood mononuclear cells (PBMC) from both wild-type (WT) and GT-KO pigs by flow cytometry. Complement-dependent cytotoxicity was measured in 39 sera. Antibody and cytotoxicity responses were measured in two baboons exposed to a GT-KO pig heart, one not immunosuppressed and one that received only cobra venom factor.IgM and IgG bound to 95% and 79% of WT PBMC, and 32% and 9% GT-KO PBMC, respectively (WT vs. GT-KO, P < 0.01). Whereas 97% of sera were cytotoxic to WT PBMC, only 64% were cytotoxic to GT-KO PBMC, and the level of cytotoxicity was less (mean 60% vs. 25% lysis, P < 0.05). In the two baboons exposed to GT-KO hearts, anti-non-Gal antibodies increased markedly, peaking after 2 (IgM) and 3 (IgG) weeks, associated with an increase in lysis of GT-KO PBMC.Two-thirds of baboon sera demonstrated cytotoxicity to GT-KO PBMC. After GT-KO organ transplantation, if an elicited antibody response develops, it is likely to cause rapid graft rejection.