In the present study we investigated whether peripheral blood gene expression measurements may serve as an early and non-invasive tool to predict renal allograft rejection. Peripheral blood was collected twice weekly after transplantation and gene expression was measured using real-time polymerase chain reaction (PCR). Recipients with acute rejection (n = 17) had higher levels of perforin and granzyme B transcript on days 5–7, 8–10, 11–13, 17–19, 20–22, and 26–29, as compared to patients without rejection (n = 50, p < 0.05 in all cases). Rejection diagnosis using gene expression criteria, determined with receiver operating characteristic (ROC) curves, was possible 2–30 days before traditional diagnosis (median 11 days). The best diagnostic result was obtained from samples taken on days 8–10, with a specificity of 90% and a sensitivity of 82% for perforin, and a specificity of 87% and sensitivity of 72% for granzyme B. Decreases in perforin (p < 0.01) and granzyme B expression (p < 0.05) were observed after initiation of anti-rejection therapy. Our data indicate that gene expression measurement is a useful tool for the recognition of graft rejection in its earliest stages. Serial measurements could be implemented as a monitoring system to highlight patients at higher risk of rejection, making them candidates for biopsy or pre-emptive anti-rejection therapy.