Urinary monitoring of kidney allograft function has been used for many years. More recently, molecular identification of cytotoxic T-cell products has been used as a diagnostic tool in acute rejection. Monitoring of T-cell infiltrates by analysis of the T-cell receptor (TcR) gene usage has been performed on biopsies with acute and chronic rejection, but not on urine samples. The aim of this study was to identify and compare TRBV gene usage assessing the CDR3 (Complementarity Determining Region 3) length distribution and sequence in urine and biopsies of pediatric renal allograft patients at the time of acute rejection and compare them with peripheral blood. We studied four pediatric renal transplant recipients with acute cellular rejection. We identified restricted and matched TRBV CDR3 spectratypes with overexpressed TRBV families and show identical, clonally expanded TRBV CDR3 sequences in all four patients present in the urine and renal allograft. We demonstrate that urinary monitoring can detect graft-infiltrating lymphocytes in acute rejection and may have a role in the monitoring of renal transplants.