OBJECTIVE - The objective of this study was to determine whether tolerance to neonatal porcine islet (NPI) xenografts could be achieved by short-term administrations of anti-LFA-1 and anti-CD154 monoclonal antibodies (mAbs). RESEARCH DESIGN AND METHODS - Diabetic B6 mice received NPI transplants and short-term injections of combined anti-LFA-1 and anti-CD154 mAbs. Mice with long-term islet graft function were treated with depleting anti-CD25 mAb or retransplanted with a second-party NPI. At the end of the study, grafts from mice with long-term islet function were examined. Their spleen cells were characterized and used for in vitro proliferation and adoptive transfer studies. RESULTS - All mAb-treated NPI recipients maintained normoglycemia for >100 days post-transplantation. Only 5 of 50 mice rejected their grafts before 300 days post-transplantation. Intact islets, foxp3+ immune cells, as well as interleukin (IL)-10 and transforming growth factor (TGF)-β regulatory cytokine transcripts were detected in the NPI xenografts from tolerant mice. A higher percentage of CD4+ T-cell population from these mice expressed regulatory markers, suggesting that tolerance to NPI xenografts may be mediated by T regulatory cells. This was confirmed when tolerant mice treated with depleting anti-CD25 mAb became diabetic. Lymphocytes from tolerant mice inhibited the proliferation of lymphocytes from B6 mice immunized with porcine cells and they displayed limited proliferation when adoptively transferred. All protected B6 mice transplanted with a second-party NPI xenograft maintained long-term normoglycemia even after removal of the first NPI graft-bearing kidney. CONCLUSIONS - These results demonstrate that tolerance to NPI xenografts can be achieved by transient administrations of combined anti-LFA-1 and anti-CD154 mAb therapy.