The present study aimed to evaluate the effect of depletion of macrophages and/or natural killer (NK) cells on islet xenograft rejection in the pig-to-mouse model. Five microliters (4,000 to 5,000 IEQ, islet equivalents) of adult pig islets were transplanted under the renal capsule of C57BL/6 mice with streptozotocin-induced diabetes. Macrophages were depleted by injection of liposome-encapsulated dichloromethylene diphosphonate (Lip-Cl2MDP) intraperitoneally (i.p.) at a dose of 100 microl/ 10 g body weight (BW) 2 days before transplantation, and 50 microl/10 g BW weekly thereafter. NK cells were depleted by injection of the monoclonal antibody NK 1.1 (anti-NK 1.1 mAb) i.p. at a dose of 100 microg/mouse 1 day before transplantation, and then 25 microg per week thereafter. Islet graft survival was monitored by daily measurements of blood glucose. Graft survival was 8 +/- 1.2 days in untreated controls, 9 +/- 1.0 days with anti-NK 1.1 mAb alone, 22 +/- 4.9 days with Lip-Cl2MDP alone (P<0.01 vs. controls), and 26 +/- 3.8 days with Lip-Cl2MDP plus anti-NK 1.1 mAb (P<0.01 vs. controls). In the last group, two of six animals were killed with functioning grafts 30 days after transplantation. In untreated controls, rejected xenografts were heavily infiltrated by F4/80+ macrophages and CD3+T cells. In Lip-Cl2MDP-treated groups, the number of F4/80+ macrophages was markedly reduced. On the periphery of xenografts, a small number of CD3+T cells were observed. In conclusion, our results suggest that strategies targeting macrophages may facilitate islet xenograft survival. A role for NK cells cannot be excluded, but appears to be of minor importance.