The success of the new immunosuppressive regime known as the Edmonton protocol in islet allotransplantation may suggest that it is also possible that this regime may prevent the rejection of xenografts. This protocol applies a combination of Tacrolimus, Sirolimus and Daclizumab at low doses. This combination may have some toxicity that affects the function and viability of the pancreatic islets. The choice of species or age category, whose islets can tolerate the toxicity of this immunosuppressive combination, may become important for the graft survival. It was the aim of this study to investigate the long-term effect of this regime on insulin secretion from pancreatic islets isolated from two species (rats and pigs) and from two age categories (day 7 postnatal [P7] and adult rat). Islets were cultured for three weeks in medium containing Tacrolimus in a concentration of 5 ng/ml, while the concentration of Sirolimus was 15 ng/ml. Daclizumab was added at the beginning of culture and once weekly in a final concentration of 10 ng/ml. In immunosuppressive-treated groups, Glucose was able to stimulate increases of insulin secretion over the basal value after 1 and 3 weeks in adult rat islets, and could not stimulate this secretion in P7 islets, while it stimulated the secretion only after 1 week, but not 3 weeks, in porcine islets. The immunosuppressive regimen caused significant reductions of glucose-stimulated insulin secretion magnitude in adult rat and porcine islets after 3 weeks, while it reduced both basal and stimulated secretions after 1 and 3 weeks in P7 rat islets. There was no difference in DNA contents between control and immunosuppressive-treated groups after 1 or 3 weeks in any of the islet preparations. DNA decreased considerably with the time in culture. The change in DNA content over 3 weeks was higher in the Edmonton group of adult porcine and P7 rat than in adult rat islets. Comparison of the responses of islets from different age categories and species leads to conclude that in vitro cultures of adult rat islets are more tolerant to this immunosuppressive combination toxicity than P7 islets, and there is variable responses of islets from different adult species to this toxicity.