Background. "Infectious tolerance" has been defined as the tolerance induced in a new recipient by the adoptive transfer of cells from a recipient accepting an allograft after anti-CD4 and anti-CD8 monoclonal antibody treatment. A clear understanding of the mechanisms responsible for graft acceptance after donor-specific blood transfusion (DST) has remained elusive. We examined the development and "infectious" nature of immunologic changes resulting in indefinite survival of LEW to DA rat cardiac allografts after DST alone without the need for antibody. Methods. One hundred × 106 LEW splenocytes (SC) as DST were injected intravenously into DA recipients 7 days before LEW cardiac transplantation. Subsequently, 100 × 106 SC harvested from a DA recipient 30, 60, or 100 days after graft acceptance were adoptively transferred into lightly γ-irradiated (450 rad) naïve DA recipients 24 hours before a second LEW cardiac allograft. Subsequent graft function was determined. Results. Adoptive transfer of SC from the DST-treated DA rats 30 days after LEW heart transplant acceptance into naïve γ-irradiated DA rats failed to transfer tolerance to LEW cardiac allografts. However, SC from DA rats bearing LEW hearts for more than 60 days induced indefinite tolerance to all LEW hearts. This infectious tolerance could be adoptively transferred again to a second DA recipient. Conclusions. DST-generated regulatory cells can down-regulate naïve lymphocytes to promote allograft acceptance. This tolerance can be expanded and serially transferred to a subsequent naïve cardiac recipient.