To evaluate whether further improvement in porcine islet xenotransplantation is feasible, a number of questions were addressed. Earlier we showed significant improvement in the nude mouse of the porcine islets by selection through long-term culture. Now these islets were tested in the stringent pig-to-rat model. Islets were isolated from adult pigs, cultured for 1.5-3 weeks and transplanted to rats. Possible rejection mechanisms were assessed by interference of the cellular response with cyclosporine A (CsA), blocking macrophages with gadolinium chloride (GdCl), and suppressing the humoral response with cyclophosphamide. Modifications in graft size and condition were analyzed. Untreated control recipients showed primary nonfunction (PNF). CsA treatment could fully overcome PNF and resulted in graft survival from 10 to over 134 days. Rejection was the main cause of function loss. Although rejection could not be prevented by intensifying the induction therapy, increased maintenance immunosuppression effectively blocked rejection, albeit at the expense of toxicity. Blocking the humoral response was ineffective; all grafts showed PNF. In contrast, depletion of macrophages fully prevented PNF. Combination of GdCl and CsA gave no additional effect, and grafts were rejected between 57 and 162 days. Generally, graft survivals were similar to those reported in the literature; however, long-term cultured islets required much less maintenance immunosuppression. Cessation of graft function was not always due to rejection; in some cases "islet exhaustion" was found, possibly caused by discrepancy between the graft size and the rapidly growing recipient. Neither the presence of damaged islet tissue in the graft nor the size of the graft exerted any influence on graft survival. On rejection, no real infiltration of the graft was seen; destruction gradually processed from the outside. The good functional capability of the cultured islets was illustrated by disappearance of the clinical symptoms and increase in body weight, which almost doubled in the long-term survivors.