Oxygenation defects may contribute to renal disease but pO2 is difficult to measure. Microelectrodes offer quantitative but localised measurements and are invasive. A global “map” of pO2 can be obtained post-mortem by immunohistochemical detection of pimonidazole adducts. This is non-quantitative and insensitive. BOLD MRI provides an alternative for longitudinal measurement of oxygen gradients. However, post-acquisition data analysis often relies on manual selection of region(s) of interest, which excludes from analysis significant quantities of biological information and is subject to selection bias. We applied k-means clustering as an anatomically unbiased analytical approach, clustering individual voxels on quantitative nearness of the R2* signal. This quantitative similarity does not necessarily imply close anatomical association of voxels or compartmentalization within a given region of the kidney. Scans were performed on anesthetized (2% Isofluorane) F344 rats (n=6) using a 7T MRI scanner. A birdcage volume coil and a 4-channel phased array surface coil were used for radio frequency transmission and signal reception, respectively. We employed two complementary strategies to affect R2*. Acutely, we administered acetylcholine IV; chronically we infused via osmotic minipump angiotensin II (60ng/min) over a 3-day period, after which rats were rescanned. Data (mean ± SE) were analysed by RANOVA. In control rats, low R2* clustering was located predominantly within the cortex and higher R2* clustering within the medulla (70.96±1.48 versus 79.00±1.50; 3 scans per rat; n=6; P<0.01) consistent anatomically with a cortico-medullary oxygen gradient. An IV bolus of acetylcholine caused a transient reduction of the R2* signal in both clustered segments (P<0.01), which was nitric oxide dependent. Chronic infusion of angiotensin II disrupted the cortico-medullary gradient, producing less distinctly segmented mean R2* clusters (71.30±2.00; versus 72.48±1.27; n=6; NS). The acetylcholine-induced attenuation of the R2* signal was abolished by chronic angiotensin II infusion, consistent with reduced nitric oxide bioavailability. In summary, we have developed an anatomically unbiased method for the assessment of renal function by BOLD MRI, employing signal analysis to remove errors inherent in manual ROI selection. This method offers a global insight into intrarenal oxygenation and the dynamic response of R2* to acetylcholine can provide information relating to renal NO bioavailability.