© 2014 Elsevier B.V. Background: Mixed chimerism is associated with donor-specific tolerance. Spleen or splenocyte allotransplantation (Tx) is recognized as potentially tolerogenic. There is no definitive report comparing chimerism levels following spleen and splenocyte Tx in a large animal model. We have compared chimerism after spleen, splenocyte, or kidney Tx in pigs. Methods: Outbred (n. =. 5) and MHC-defined miniature (n. =. 1) pigs underwent orthotopic spleen Tx. Outbred pigs received splenocytes through a systemic vein (n. =. 1) or the portal vein (n. =. 3). Kidney Tx (n. =. 2) or concomitant Tx of spleen. +. kidney (n. =. 2) was carried out. All except one recipient pigs were irradiated (700. cGy thymic and 100-125. cGy whole body) on day. -. 2. Cyclosporine or tacrolimus was administered for 42. days. All donors were males and all recipients were females; chimerism in the blood was determined by Quantification-PCR for the donor Y chromosome. Mixed lymphocyte reaction (MLR) was performed before and after Tx. Results: One week after spleen Tx in outbred and MHC-defined pigs, chimerism ranged between 0.8 and 22.5%, and 5.4-20.1%, respectively, and remained between 17.7 and 67.4%, and 2.2-7.4%, respectively, until day 28. One week after splenocyte Tx, chimerism ranged between 0.1 and 8.5%, and decreased to 0.1-0.8% at 3-4. weeks. There was no detectable chimerism 14. days after kidney Tx. The response on MLR of all recipient pigs to donor cells was decreased after Tx, except in one case of splenocyte Tx, indicating that this pig might have become sensitized. After discontinuation of immunosuppression, most isolated spleen or kidney grafts were not rejected, but the kidney was rejected after concomitant spleen. +. kidney Tx. Conclusions: There was a significantly higher level of blood chimerism following spleen Tx compared to splenocyte or kidney Tx. However, concomitant Tx of spleen. +. kidney may be associated with accelerated kidney graft rejection.