Non-donor-specific cardiac allograft acceptance is induced in C3H/He (C3H; H-2 k ) recipients injected as neonates with allogeneic BALB/c (BALB; H-2 d ) fetal liver cells (FLC). This occurs despite intact reactivity to donor-type and third-party alloantigens in in vitro assays and skin transplants. To investigate a role for regulatory T cells, we performed adoptive transfer studies and specifically assessed CD4 + and CD4 − T cells. Three cell populations (splenocytes, CD4 + , CD4 − ) derived from neonatally-treated mice with accepted C57BL/6 (B6; H-2 b ) third-party cardiac grafts were adoptively transferred into sub-lethally-irradiated C3H mice. Reconstituted mice were challenged with B6 cardiac grafts, B6 skin grafts, or unrelated cardiac grafts. Separated cells were assessed in vitro. B6, BALB, and NZW (H-2 z ) graft acceptance was transferred by unfractionated splenocytes. CD4 + cells transferred B6 graft acceptance (85% survival > 100 days). CD4 − cells, unfractionated cells from naive or only irradiated mice, and unfractionated cells from neonatally-treated non-transplanted C3H mice rejected grafts within 35 days. No inoculum induced skin graft acceptance. Co-cultured assays confirmed the suppressive function of CD4 + cells in vitro. Cardiac allograft acceptance in our model is regulated by CD4 + cells. The regulatory cell population is induced by the cardiac graft itself and mediates in vivo cardiac graft acceptance in a tissue-specific but not donor-strain-specific manner.