In human organ transplantation, the correct diagnosis of rejection may be difficult using clinical and/or histopathologic criteria, and immunologic assays should therefore be considered. We have applied monoclonal antibodies in 2-color flow cytometric (FACS) analysis to study phenotypic patterns of kidney-infiltrating activated and functional T and NK cell subsets in 132 posttransplantation biopsies. Viable intragraft lymphocytes and kidney tubular cells were obtained by the use of medium-sized ultra-sound guided needle biopsy. In univariate analyses, highly significant differences were observed between rejecting and stable grafts. During rejection, the proportions of HLA-DR+ kidney tubular cells, total lymphocytes, T suppressor/cytotoxic cells, and natural killer (NK)-like cells increased; the fractions of phenotypically activated, HLA-DR+ cells within the latter 2 cell populations were elevated. Further, during rejection phenotypic T suppressor inducer and T suppressor effector cells were virtually absent in kidney tissue but reappeared rapidly upon successful antirejection therapy. Multivariate analyses revealed that combinations of antibodies in FACS analysis defining functional and activated subsubsets of T and NK cells allow a precise and rapid immunologic diagnosis of kidney rejection.