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Objectives Morphological imaging techniques are performing poorly in assessing the response to preoperative radiotherapy, mainly because of desmoplastic reactions. The aim of this study was to investigate the potential of sequential 18FDG-PET in assessing the response of rectal cancer to neo-adjuvant radiotherapy (RT), and to determine which parameters can be used as surrogate markers for histopathological response. Methods 18FDG-PET scans were acquired prior to and in the 5th week after the end of RT. Tracer uptake was assessed semi-quantitatively using standardized uptake values (SUV). The percentage difference (%Δ) between pre- and post RT scans in SUVmax, SUVmean, metabolic volume (MV) and the total glycolytic volume (tGV) were calculated. Results Forty-five consecutive patients with histologically confirmed rectal adenocarcinoma were enrolled. Following neo-adjuvant RT, of the 45 patients 20 were classified as histopathological responders and 25 as non-responders. Intense 18F-FDG uptake was seen inall tumors prior to neo-adjuvant radiotherapy (average SUVmax 12.9±6.0). When classifying patients in histological responders and non-reponders. Significant differences in %ΔSUVmax (55.8% vs 37.4%, p=0.023) and %ΔSUVmean (40.1% vs 21.0%, p=0.001) were observed between both groups. For %ΔMV and %ΔtGV decreases were more prominent in responders, but not significantly different from non-responders. As demonstrated by ROC analysis %ΔSUVmean was a more powerful discriminator than %ΔSUVmax. The sensitivity, specificity, accuracy, positive and negative predictive value for optimal threshold of %ΔSUVmean (24.5%) was 80, 72, 76, 70 and 82% respectively. Conclusions Sequential 18FDG-PET allows assessing the response to preoperative radiotherapy. Both %ΔSUVmean and %ΔSUVmax correlate with histopathological response