Control of intestinal iron absorption is crucial for body iron homeostasis. We have shown that short-term (3 d) hypoxia promotes iron uptake across the duodenal brush border membrane (O’Riordan et al. 1997). This present study reports the effect of prolonged hypoxia on mucosal iron uptake as well as iron transfer to the blood and the expression of hepcidin, a proposed negative regulator of duodenal iron transport (Laftah et al. 2004). Sprague-Dawley rats (90-100 g) were exposed to 10% oxygen or room air for 28-33 d. During this time they were pair-fed and weighed weekly. Before experimentation, animals were terminally anaesthetized (pentobarbitone sodium, 90 mg.kg, i.p) and blood and liver samples were taken to determine haemoglobin (Hb) level and gene expression of hepcidin respectively (Laftah et al. 2004). For mucosal uptake and autoradiographic studies, 2 cm everted duodenum was pre-incubated for 5 min in oxygenated, well-stirred Hepes buffer containing 10 mM glucose (O’Riordan et al. 1997). The tissue was then exposed for 5 min to this buffer with added 0.2 mM 59Fe2+:ascorbate (pH 6) and then washed free of surface bound iron and fixed. Tissues were weighed, gamma-counted and processed for autoradiography (O’Riordan et al. 1997). Parallel in vivo experiments on rats under terminal anaesthesia (pentobarbitone sodium, 90 mg.kg, i.p) measured the rate of appearance of 59Fe in blood for <30 min after instillation of buffer containing 0.2 mM 59Fe2+:ascorbate into closed 5 cm duodenal loops (O'Riordan et al. 1995). Rats were then humanely killed. Hypoxia increased Hb concentration by 47.2% (means±s.e.m.: 12.7±0.4 (n=5) vs 18.7±0.6 (7) g/100 ml p<0.001, Students unpaired t-test), and reduced the expression of liver hepcidin mRNA by 60% (2.70±0.56 (3) vs 1.08±0.34 (4) arbitrary absorbance units, p<0.05). Hypoxia promoted both mucosal iron uptake (70.6±6.0 (6) vs 206.6±57.5 (7) pmoles/g tissue, p<0.05) and lumen to blood iron transfer (20 min: 0.60±0.10 (8) vs 1.14±0.20 (8), 30 min: 0.70▒0.11 [8] vs 1.92▒0.50 (8) pmoles/ml blood/g mucosa, both p<0.05). Hypoxia did not affect villus length but autoradiographic analysis showed that this condition increased enterocyte 59Fe content at all stages of cell maturation, particularly the mid-villus region, without affecting the overall developmental profile of villus iron uptake. The function of these responses is to provide a higher level of body iron to satisfy the requirements for increased erythropoiesis during hypoxia. Reduced secretion of hepcidin is likely to be pivotal to the adaptive changes in uptake, but the processes leading to suppression of hepcidin expression during hypoxia are unknown.