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 post-transplantation biopsies. Viable intragraft lymphocytes and kidney tubular cells were obtained by the use of medium-sized ultra-sound guided needle biopsy. In uni-variate 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 antirejec-tion 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.