BACKGROUND The development of a noninvasive method to diagnose renal allograft rejection could prevent the complications associated with graft biopsy and allow more accurate surveillance of allograft function. The present study determines whether expression of CD69 on peripheral T lymphocytes of renal allograft recipients correlates with the presence of acute graft rejection. METHODS Peripheral blood T lymphocytes from healthy volunteers, renal allograft recipients with elevated creatinine but no evidence of rejection on biopsy, and renal allograft recipients with biopsy-proven rejection were analyzed by flow cytometry for the expression of CD69 and various intracellular cytokines (interleukin-2, interferon-gamma). Results were then compared with the degree of rejection on biopsy. RESULTS CD69 expression on CD3+, CD4+, and CD8+ T-cell subsets was low in controls and transplant recipients without allograft rejection. In contrast, patients with renal allograft rejection showed significantly elevated percentages of CD69+ cells in the CD3+ (P<0.01) and CD8+ subsets (P<0.01). The fraction of CD69+ and CD8+ T cells was found to be a more clinically useful test based on receiver-operator characteristics. CD69 expression on CD4+ T cells did not correlate with rejection. Significant intracellular cytokine levels were not detected in unstimulated T cells from any of the groups; stimulation with mitogens increased expression equally among the three groups. CONCLUSIONS We demonstrate that expression of CD69 on CD3+ and CD8+ peripheral blood T cells correlates closely with the presence of acute graft rejection in renal allograft recipients. Measurement of this surface marker may provide a rapid, noninvasive, and accurate means by which graft rejection can be identified.