The hypoxia-mediated response of tumours is a major determining factor in growth and metastasis. Understanding tumour biology under hypoxic conditions is crucial for the development of antiangiogenic therapy. Using one of the largest cohorts of rectal adenocarcinomas to date, this study investigated hypoxia-inducible factor-1alpha (HIF-1alpha) and HIF-2alpha protein expression in relation to rectal cancer recurrence and cancer-specific survival. Patients (n=90) who had undergone surgery for rectal adenocarcinoma, with no prior neoadjuvant therapy or metastatic disease, and for whom adequate follow-up data were available were selected. Microvessel density (MVD), HIF-1alpha and HIF-2alpha expressions were assessed immunohistologically with the CD34 antibody for vessel identification and the NB100-131B and NB100-132D3 antibodies for HIF-1alpha and HIF-2alpha, respectively. In a multifactorial analysis, results were correlated with tumour stage, recurrence rate and long-term survival. Microvessel density was higher across T and N stages (P<0.001) and associated with poor survival (hazard ratio (HR)=8.7, P<0.005) and decreased disease-free survival (HR=4.7, P<0.005). hypoxia-inducible factor-1alpha and -2alpha were expressed in >50% of rectal cancers (HIF-1alpha, 54%, 48/90; HIF-2alpha, 64%, 58/90). HIF-1alpha positivity was associated with both TNM stage (P<0.05) and vascular invasion (P<0.005). In contrast, no associations were demonstrated [corrected] between HIF-2alpha [corrected] and any pathological features or [corrected] outcome. The study showed an independent association between HIF-1alpha expression and advanced TNM stage with poor outcome. Our results indicate that HIF-1alpha, but not HIF-2alpha, might be used as a marker of prognosis, in addition to methods currently used, to enhance patient management.