Our aim is to develop noninvasive tests to monitor the renal allograft posttransplant. Previously, we have reported that an unbiased proteomic-based approach can detect urine protein peaks associated with acute tubulointerstitial renal allograft rejection. Identification of these proteins peaks by mass spectrometry demonstrated that they all derive from nontryptic cleaved forms of beta2-microglobulin. In vitro experiments showed that cleavage of intact beta2-microglobulin requires a urine pH < 6 and the presence of aspartic proteases. Patients with acute tubulointerstitial rejection had lower urine pH than stable transplants and healthy individuals. In addition, they had higher amounts of aspartic proteases and intact beta2-microglobulin in urine. These factors ultimately lead to increased amounts of cleaved urinary beta2-microglobulin. Cleaved beta2-microglobulin as an indicator of acute tubular injury may become a useful tool for noninvasive monitoring of renal allografts.