Neonatal skin allografts can be tolerogenic when transplanted to appropriately immunosuppressed hosts. Single grafts of neonatal skin survive longer than adult skin grafts when recipients are treated with antilymphocyte serum (ALS) and donor bone marrow cells (BMC). Neonatal skin grafts can also prolong the survival of adult grafts of the same donor strain simultaneously cotransplanted with the neonatal grafts. To probe the mechanisms involved in this cotransplantation phenomenon, we delayed placement of the neonatal cotransplants relative to grafting with adult skin. Neonatal allografts placed either 7-9 days or 14 days after grafting with adult skin significantly prolonged adult graft survival in mice treated with ALS and BMC. However, day 0-placed neonatal cotransplants must remain on the recipient for > 2 weeks to prolong adult graft survival. Removal of cotransplants from ALS- and BMC-treated recipients after 7 or 14 days abrogated the cotransplantation effect. If left in place until day 21, neonatal cotransplants could significantly prolong adult graft survival, but did not induce the long-term graft survival observed in approximately 50% of the recipients whose cotransplants were not removed. Cotransplant removal after 1 year did not affect subsequent adult graft survival. Additionally, cotransplants were removed from recipients either on day 14 or from long-term graft-bearing mice and retransplanted to other ALS/BMC-treated recipients. These retransplanted grafts were unable to prolong survival of adult grafts on the new recipients. After transplant, but not before transplant, cyclophosphamide treatment of recipients prevented expression of the cotransplant effect in ALS-treated mice. However, recipient splenectomy > or = 1 week before grafting did not interfere with the effect. These results reflect on the contributions of the donor tissue, and the recipients' response, to the tolerogenic signals that permit a neonatal cotransplant to prolong adult graft survival.