Urine cytology (UC) has proved to correlate well with core and fine-needle aspiration kidney biopsies of renal allograft recipients undergoing acute rejection (AR). This study was undertaken to compare the relative usefulness of urine flow immunocytometry (UFC) (using fluorescinated antibodies anti-HLA-DR, anti-CD3 and antirenal epithelial cells) with UC in its ability to diagnose AR by analyzing 200 urine specimens during a prospective double-blind study of 40 renal transplant recipients. Clinical diagnosis was retrospectively assigned to one of the following categories: group I--AR, 15; group II--ischemic injury period (first 5 days postop.), 12; group III, 173 (including 168 stable grafts, 1 pyelonephritis and 4 cyclosporine toxicity), by investigators blinded to the urine results. Both tests were highly sensitive for the diagnosis of AR (UC = 86.6% vs. UFC = 100%; P = NS) with a specificity after the ischemic injury period of 78% by UC and 87.9% by UFC. Samples obtained during AR revealed higher levels of expression of HLA-DR as well as higher numbers of CD3-positive cells. These tests had specificity values of 95.3% and 97.6%, respectively, for the diagnosis of AR. The degree of immune activation (established by numbers of lymphocytes/lymphoblasts seen by UC) correlated with the severity of biopsy-proven ARs and with response to antirejection therapy. In conclusion, both test are highly accurate in diagnosing AR. The highest specificity value was obtained when both UC and UFC were utilized together (93%). We suggest that the routine use of these tests can provide an important adjunct to the evaluation of renal transplant recipients.