Hypoxia-associated markers are involved in the progression of several malignancies, but are relatively unstudied in Barrett's carcinogenesis. Our aim was to assess the immunohistochemical expression of hypoxia-inducible factor (HIF)-1α, HIF-2α, erythropoietin (Epo), Epo receptor (Epo-R), Glut-1 and vascular endothelial growth factor (VEGF) along with Ki67/MIB-1 in the Barrett's metaplasia–dysplasia–adenocarcinoma sequence. Endoscopic biopsies of normal squamous epithelium (NSE)(n= 20), columnar-lined oesophagus (CLO)(n= 15), CLO with intestinal metaplasia (n= 20), dysplasia (n= 17) and Barrett's type adenocarcinoma (n= 20) were obtained. Immunohistochemistry was performed on the paraffin-embedded tissue. A score was calculated for each marker (range 0− 300) by multiplying intensity (none 0, weak 1, moderate 2, strong 3) by percentage of expression (range 0–100). Significant increases in the expression of HIF-2α (P= 0.014), VEGF (P< 0.0001), Epo-R (P< 0.0001) and Ki67 (P< 0.0001) were found as tissue progressed from NSE to adenocarcinoma. HIF-2α was expressed late in the sequence and was only seen in dysplasia and adenocarcinoma. High HIF-2α expression was seen in 12 out of 20 Barrett's type adenocarcinoma. The late expression of HIF-2α in the Barrett's carcinogenesis sequence and its high expression in adenocarcinoma suggest that it is worth further investigation as a marker of disease progression and therapeutic target.