The effect of cyclosporin-A, low-temperature culture, and anti-Ia antibodies on prevention of rejection of rat islet allografts was determined. Wistar-Furth islets were isolated by the collagenase technique and transplanted via the portal vein into diabetic Lewis recipients. Cyclosporin-A (30 mg/kg) injected at 0, 1, and 2 days after transplantation produced a significant prolongation of survival of the islet allografts (MST > 35.7 ± 7.0 days) when hand-picked donor islets were used, whereas only a modest prolongation of survival (14.0 ± 1.6 days) was obtained using donor islets removed directly from Ficoll gradients. This difference in survival was apparently due to the large number of lymphoid, antigen-presenting cells that were present in the islet fraction removed directly from the Ficoll gradients. Treatment of donor, hand-picked islets with a mixture of cross-reactive anti-Ia antibodies and complement without cyclosporin-A therapy did not prolong the survival of islet allografts (MST, 6.5 ± 0.4 days versus 7.0 ± 0.5 days in controls). In contrast, treatment of the donor islets with the mixture of anti-Ia antibodies and complement in conjunction with the 3-day course of cyclosporin-A therapy produced an 83% survival of the islet allografts at 60 days after transplantation. In vitro culture of hand-picked donor islets at 24° C for 7 days and the 3-day course of cyclosporin-A therapy produced a 100% survival of the allografts at 60 days after transplantation. These findings indicate that rejection of rat islets transplanted into a strain with strong immune responsiveness (Lewis) can be prevented by a combination of temporary immunosuppression of the recipients with cyclosporin-A and pretreatment of the donor islets with either cross-reactive anti-Ia antibodies or culture of the islets at 24°C for 7 days before transplantation .