Background.The authors previously showed that intratracheal delivery (ITD) of donor splenocytes induced prolonged survival of fully allogeneic cardiac grafts in mice. In this study, this treatment protocol was combined with blockade of the CD40 pathway in an attempt to induce operational tolerance.Methods.CBA mice were given donor splenocytes (1×107) or Kbpeptide (100 &mgr;g) by ITD with or without antibody specific for mouse CD40 ligand (MR1, 200 &mgr;g) 7 days before transplantation of a C57BL/10 heart. Also, splenocyte (5×107) from primary recipient CBA mice given ITD of donor splenocytes or Kbpeptide plus MR1 were adoptively transferred into naive CBA secondary recipients 7 days after the pretreatment and C57BL/10 hearts were transplanted into those recipients the same day.Results.ITD of donor splenocytes and Kbpeptide induced prolonged survival of cardiac grafts (median survival time [MST], 74 and 56 days, respectively), whereas naive control mice and mice pretreated with syngeneic splenocytes had acute graft rejection (MST in both groups, 7 days). When MR1 was included, all grafts survived indefinitely (>200 days), but mice pretreated with MR1 alone had graft rejection (MST, 54 days). Mice bearing cardiac grafts had acceptance of skin grafts from C57BL/10 but not BALB/c mice, demonstrating that operational tolerance was induced. Secondary recipients given adoptive transfer of splenocytes from primary recipients of the combined treatment had acceptance of C57BL/10 grafts, suggesting that regulatory cells were generated within 7 days of pretreatment.Conclusions.ITD of donor splenocytes or Kbpeptide under blockade of the CD40 pathway induced operational tolerance and generated regulatory cells.