Flow and oxygenation dependent (FLOOD) MR images of GH3 prolactinomas display large intensity increases in response to carbogen (5% CO2/95% O2) breathing. To assess the relative contributions of carbon dioxide and oxygen to this response and the tumour oxygenation state, the response of GH3 prolactinomas to 5% CO2/95% air, carbogen and 100% O2 was monitored by FLOOD MRI and PO2 histography. A 10-30% image intensity increase was observed during 5% CO2/95% air breathing, consistent with an increase in tumour blood flow, as a result of CO2-induced vasodilation, reducing the concentration of deoxyhaemoglobin in the blood. Carbogen caused a further 40-50% signal enhancement, suggesting an additional improvement due to increase blood oxygenation. A small 5-10% increase was observed in response to 100% O2, highlighting the dominance of CO2-induced vasodilation in the carbogen response. Despite the large FLOOD response, non-significant increases in tumour pO2 were observed in response to the three gases. Tissue pO2 is determined by the balance of oxygen supply and demand, hence increased blood flow/oxygenation may not necessarily produce a large increase in tissue PO2. The FLOOD response is determined by the level of deoxygenation of blood, the size of this response relating to vascular density and the potential of high-oxygen content gases to improve the oxygen supply to tumour tissue.