The immunomodulatory enzyme indoleamine 2,3-dioxygenase (IDO) is activated by interferon-gamma (IFN-gamma) and via tryptophan depletion, suppresses adaptive T cell-mediated immunity in inflammation, host immune defense, and maternal tolerance. Its role in solid organ transplantation is still unclear. Therefore, we investigated the usefulness of IDO-mediated tryptophan catabolism in the evaluation of kidney allograft rejection. Blood, urine, and tissue samples were collected from 34 renal transplant patients without rejection and from nine patients with biopsy-confirmed episodes of acute rejection (n=12). Concentrations of kynurenine and tryptophan in serum and urine were analyzed by high-pressure liquid chromatography. Kynurenine to tryptophan ratio (kyn/trp) was calculated to estimate IDO activity. Immunostaining for IDO was performed on renal biopsies. Neopterin was assessed using radioimmunoassay. Kyn/trp and neopterin were detectable at low levels in serum of healthy volunteers and were increased in non-rejecting allograft recipients. Serum levels of kyn/trp were higher in recipients with rejection compared to non-rejectors as early as by day 1 post-surgery. Rejection episodes occurring within 13+/-5.9 days after transplantation were accompanied by elevated kyn/trp in serum (114+/-44.5 micromol/mmol, P=0.001) and urine (126+/-65.9 micromol/mmol, P=0.02) compared to levels during stable graft function. Kyn/trp correlated significantly with neopterin suggesting an IFN-gamma-induced increase in IDO activity. Immunostaining showed upregulation of IDO in rejection biopsies, localized in tubular-epithelial cells. Non-rejected grafts displayed no IDO expression. Acute rejection is associated with simultaneously increased serum and urinary kyn/trp in patients after kidney transplantation. Thus, IDO activity might offer a novel non-invasive means of immunomonitoring of renal allografts.