The effect of long-term pharmacologic immunosuppression (PI) on anti- Galα1-3Gal (proportional to Gal) antibody (Ab) levels has not been determined previously in humans. In this study, we measured proportional to Gal Ab levels by ELISA in 14 healthy volunteers (controls) and in 70 patients with grafts (kidney, heart, liver) who had received different combinations of PI (including cyclosporine, tacrolimus, azathioprine, mycophenolate mofetil, and steroids) for >3 months. There was great variation in Gal IgM (<80-fold) and IgG (<160-fold). There was no difference in Gal IgM or Gal IgG between any one group and any other. In kidney patients with either high (mean 68%) or low (mean 6%) panel-reactive alloantibodies, there was no difference in proportional to Gal Ab level or serum cytotoxicity to pig cells. In vitro immunoadsorption of proportional to Gal Ab from the serum did not change panel-reactive alloantibody positivity. Therapy with OKT3, a mouse product that might stimulate proportional to Gal Ab production, led to no significant change in patient Ab levels. We conclude that long-term (>3 months) PI does not reduce proportional to Gal Ab levels sufficiently to be of clinical value in xenotransplantation.