In certain combinations of donor and recipient rat strains, such as DA (RT1a) donors into PVG (RT1c) recipients, rejection after orthotopic liver transplantation (OLT) is overcome without immunosuppressive drugs, although other organs transplanted between these combinations are promptly rejected. The mechanisms involved in achieving drug-free liver allograft tolerance still remain poorly understood. In the present study, OLT (DA into PVG) serum from various postoperative times was analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis and two unique proteins of 40 kDa and 37 kDa were found to be in large concentrations in 60 day post-OLT serum. These proteins could only be detected at specific times after OLT in the DA into PVG combination and could not be detected in the serum of syngenically transplanted animals (DA into DA) and (PVG into PVG), rejector combinations (DA into LEW) requiring immunosuppressive treatment or induced by other organ transplants. When these proteins were purified and sequenced they were found to have an identical N-terminal sequence which is not listed in sequence databases. Mixed lymphocyte assays revealed that only the 40 kDa protein has a immunosuppressive capability which additionally appears to be donor specific. The 40 kDa protein will aid further in the understanding of how drug-free tolerance is attained in certain liver allografts and may also act as a marker of when treatment with conventional immunosuppressive drugs can be stopped in clinical OLT providing a homologue of the molecule can be found. This possibility appears likely as case reports already exist of patients who have successfully been able to cease treatment with such drugs.