Studies were conducted to determine the functional characteristics of lymphocytes infiltrating human heart allografts. We have developed methodologies to generate lymphocyte cultures from endomyocardial biopsies. Thirteen biopsies from four heart transplant recipients, obtained at different days during a posttransplant period of less than two months, were cultured in interleukin-2 (IL-2)-containing medium supplemented with irradiated autologous peripheral blood lymphocytes as feeder cells. Lymphocyte cultures were obtained from all 13 biopsies and they exhibited a proliferative response to IL-2, suggesting the presence of activated T cells that express IL-2 receptors. Several cultures consisted of Leu 3 (helper/inducer) T cells, whereas others were primarily Leu 2 (cytotoxic/suppressor) T cells or a mixture of both types of cells. Cultured lymphocytes were also shown to be able to undergo secondary proliferation to donor-specific leukocytes as measured by primed lymphocyte testing (PLT). The PLT specificity of these cells was frequently toward class II HLA antigens of the donor, but certain cultures had PLT specificity associated with class I HLA antigens. These results demonstrate the feasibility of growing functionally active T cells from heart transplant biopsies. An analysis of the phenotypes and allospecificity, as well as a functional characterization of these cells, should generate useful information about the types of T cells involved in cardiac transplant rejection.