The natural anti-Gal antibody seems to create a major obstacle for discordant xenotransplantation in humans. Anti-Gal, which is produced in large amounts in humans (1% of circulating IgG), interacts specifically with the carbohydrate structure Gal [alpha] l-3Gal [latin sharp s] 31-4Glc-NAc-R (termed the [alpha]-galactosyl epitope). This epitope is present in large amounts on porcine cells, as well as on cells of other nonprimate mammals (1x106 to 35x106 epitopes/cell). The interaction of anti-Gal with [alpha]-galactosyl epitopes on the xenograft was found to mediate the immune destruction of discordant xenografts. In the present study, the human immune response to [alpha]-galactosyl epitopes on xenografts was assessed by measuring changes in anti-Gal titers and affinity in sera of diabetic patients transplanted with fetal porcine islet cell clusters. The activity of this antibody was assessed by a hemagglutination assay with RBC, by ELISA with mouse laminin as a solid-phase antigen, and by equilibrium dialysis with the raolabeled free haptenic form of the [alpha]-galactosyl epitope, ie [3H] Ga1 [alpha] 1-3Ga1 [beta] 1-4G1cNAc. All assays revealed a marked increase in anti-Gal activity after transplantation. The increase in anti-Gal titers ranged between 8-and 64-fold. A similar increase was observed in the binding of free a-galactosyl epitopes to anti-Gal, as assayed in equilibrium dialysis. Immunoglobulin concentration did not increase after transplantation, suggesting that the observed increase in anti-Gal activity is the result of a specific immune response against a-galactosyl epitopes on the xenograft. The elevation in anti-Gal activity was observed in all three immunoglobulin classes and the highest activity was found within the IgG class. Analysis of IgG binding to fixed porcine endothelial cells suggested that most of the observed increased activity against these cells in transplanted patients may be attributed to the elevation in anti-Gal activity.