One-hundred consecutive human renal allograft Tru-cut needle biopsies were studied for in vitro proliferation of T lymphocytes under restrictive culture conditions containing low-dose recombinant interleukin 2. Each biopsy was entered into a blinded code and evaluated prospectively for visual evidence of growth at 24 hr and for sustained growth. Those T cell populations exhibiting sustained growth were then evaluated for cell surface phenotype by FACS; for allospecific cytotoxicity by B1Cr release; for a proliferative response to alloanti-gen by incorporation of [3H]-thymidine; and for secretion of IL-2, IL-4, IFN-7, and TNF-a in response to alloantigenic stimulation by ELISA. All results were compared with clinical diagnosis, immunosuppression at time of biopsy, diagnosis and phenotype by immuno-pathology, short-term outcome and long-term graft survival. Growth at 24 hr was predictive of acute cellular rejection (P<0.0005), unrelated to chronic rejection (P=0.663) or maintenance immunosuppression (P=0.911), and inversely correlated with cyclosporine toxicity (PsO.051) and treatment with OKT3 (P=0.014). The CD4/CD8 ratio of the sustained T cell populations was unrelated to that seen on histological examination (correlation coefficient = —0.098 and 0.044 for diffuse and aggregate infiltrates, respectively). Cytotoxic specificity for HLA class II was mediated by CD4+ cells and for HLA class I by CD8+ cells. Enhanced secretion of IL-2 in response to alloantigen distinguished those cells associated with irreversible allograft damage from those associated with complete functional recovery (P=0.01). This study demonstrates that early evaluation of T cell proliferation in vitro identifies activated T cell infiltrates mediating acute cellular allograft rejection in a time frame suitable for clinical diagnostic application. It strengthens the concept that donor-specific cytotoxicity is governed by the stabilization of the alloantigen-T-cell receptor interaction by the accessory molecules CD4 and CD8, but either interacti n is equally able to participate in an episode of acute rejection. Irreversible graft injury is associated with infiltrating cells that are capable of amplifying their responsiveness through secretion of IL-2.