Forty cyclosporine-treated renal allograft biopsies were stained with anti-Tac, a monoclonal antibody (MoAb) against interleukin 2 receptor (CD25), anti-transferrin receptor MoAb, and Ki67 MoAb (a proliferating cell marker), using a method of gold enhancement of the diaminobenzidine reaction product, in order to study the utility of these markers to differentiate rejection from other causes of graft dysfunction. Biopsies were selected on the basis of availability of sufficient frozen material and optical microscopy diagnosis, and only biopsies that showed acute cellular rejection and biopsies that did not show any sign of rejection (cyclosporine toxicity or a stable graft) were studied. In addition biopsies were stained with a panel of monoclonal antibodies reacting with CD45, CD3, CD4, CD8, EBM11 (a monocyte-macrophage marker), HLA-DR, DP, and DQ, using the usual indirect immunoperoxidase method. The clinical diagnosis (rejection versus no rejection) was made in ignorance of the biopsy findings.In 17 of 18 (94%) episodes of rejection, anti-Tac and anti—transferrin receptor—positive cells, and in 15 of 18 rejection episodes (83%), proliferating cells, were found in the interstitium. Anti-Tac—positive cells were never found in the 11 biopsies diagnosed as no rejection, and only in two a few anti—transferrin receptor—positive cells were present. The proportion of activated and proliferating cells in rejection episodes, expressed as the percentage of CD45-positive cells were: anti-Tac MoAb 4.1±2.9%, anti-transferrin receptor MoAb 6.9±3.9%, and Ki-67 MoAb 6.3±4.8%.There was a positive correlation between the total number of infiltrating cells and the number of cells stained with anti-Tac (r=0.75,P<0.005), anti-transferrin receptor MoAb (r=0.84,P<0.0001), and Ki-67 (r=0.50,P<0.05). The episodes of rejection that took place when cyclosporine levels were high had fewer 1L-2R-bearing cells in the interstitium than those that took place when cyclosporine levels were low (r=0.47,P<0.05).We conclude that during rejection the presence of activated and proliferating cells is a consistent finding, and that these markers could be useful in differentiating between rejection and other causes of graft dysfunction as well as in grading the severity of rejection.