BACKGROUND: An initial observation suggested high levels of anti-pig antibodies in healthy humans who had spent their childhood in the Middle East. We tested larger cohorts to determine whether anti-pig antibody levels correlated with the geographic location in which the subject spent his/her childhood, because this might have implications for clinical trials of xenotransplantation. METHODS: Anti-pig IgM and IgG levels (by flow cytometry using peripheral blood mononuclear cells from wild-type and α1,3- galactosyltransferase gene-knockout pigs) and anti-Gal IgM and IgG levels (by enzyme-linked immunosorbent assay) were measured in 75 volunteers. Comparisons of antibody levels were also made based on subject age, gender, ABO blood group, diet, and history of vaccination. RESULTS: Antibody binding to α1,3-galactosyltransferase gene-knockout pig cells was less than to wild-type cells. There was a reduction in anti-pig IgM and anti-Gal IgM, but a slight increase in anti-nonGal IgG, with age. Women had higher levels of anti-Gal IgM than men. Blood group A subjects had higher levels of anti-pig IgM and IgG than those of group AB. Diet had no influence on antibody levels. Typhoid or measles-mumps-rubella vaccination was associated with lower anti-nonGal IgG or anti-Gal IgG, respectively, whereas influenza vaccination was associated with higher anti-nonGal IgG. There were some significant variations in antibody levels associated with location during childhood, with subjects from the Middle East demonstrating higher anti-nonGal IgG and anti-Gal IgG. CONCLUSION: Clinical trials of xenotransplantation may be influenced by various factors, including the geographic location of the recipient during childhood, possibly associated with exposure to different microorganisms. Copyright © 2013 Lippincott Williams & Wilkins.