Limiting dilution assays to measure the frequency of interleukin-2-secreting peripheral blood T cells were carried out in patients, whose renal allografts had failed due to acute rejection (9 patients) and in patients whose grafts failed more than two years after transplantation without any recent evidence of acute rejection. Using a modified form of the assay we demonstrate that nearly half of 18 patients whose renal transplants had failed after more than two years have low or undetectable HTLp frequencies against donor, but not third-party DR antigens. No such difference was observed in any of the nine patients studied whose transplants were lost from early acute rejection. These results provide the first indication that, as in rodent models of transplantation, T cell unresponsiveness towards donor MHC antigens can occur following prolonged residence of an allograft in humans. Furthermore, the results suggest that chronic rejection may be driven by mechanisms other than direct allorecognition. The assay may be a valuable tool to study the evolution of donor-specific direct T cell alloresponsiveness in patients with well-functioning grafts.