Since the rate of immunological losses of liver allograft after the immediate posttransplant period is much lower than in other organs, we studied the immune responses against donor HLA antigens in 18 patients with a good long-term outcome to determine whether the development of a state of immunological non-responsiveness to donor antigens might account for this favorable outcome. The reactivity against donor spleen cells was measured before and 2 years after transplantation. The reactivity in mixed lymphocyte culture (MLC) and the frequencies of cytotoxic T cell precursors (CTLp) were determined. Responses against third-party spleen cells were determined concurrently to exclude a generalized reduction of immunocompetence due to chronic immunosuppressive treatment. Before orthotopic liver transplantation, the majority of patients had normal T cell responses against donor antigens that were comparable to those against third-party antigens. Two years after transplantation, donor-specific MLC non-reactivity had developed in 10 of the 18 (56%) patients. In addition, 15 of 18 (83%) patients had developed donor-specific cytotoxic T cell (CTL) non-responsiveness; 2 had reduced numbers of CTLp against both donor and third-party cells, while the remaining patient had maintained reactivity against donor antigens. In conclusion, donor-specific non-responsiveness is present in the majority of patients 2 years after successful liver transplantation, but occurs predominantly at the CTL level.