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Measurement of the cerebral metabolic rate of oxygen (CMRO2) by means of direct imaging of the 17O signal can be a valuable tool in neuroscientific research. However, knowledge of the longitudinal and transverse relaxation times of different brain tissue types is required, which is difficult to obtain because of the low sensitivity of natural abundance H217O measurements.Using the improved sensitivity at a field strength of 16.4 Tesla, relaxation time measurements in the rat brain were performed in vivo and postmortem with relatively high spatial resolutions, using a chemical shift imaging sequence.In vivo relaxation times of rat brain were found to be T1 = 6.84 ± 0.67 ms and T2* = 1.77 ± 0.04 ms. Postmortem H217O relaxometry at enriched concentrations after inhalation of 17O2 showed similar T2* values for gray matter (1.87 ± 0.04 ms) and white matter, significantly longer than muscle (1.27 ± 0.05 ms) and shorter than cerebrospinal fluid (2.30 ± 0.16 ms).Relaxation times of brain H217O were measured for the first time in vivo in different types of tissues with high spatial resolution. Because the relaxation times of H217O are expected to be independent of field strength, our results should help in optimizing the acquisition parameters for experiments also at other MRI field strengths. Magn Reson Med 75:1886–1893, 2016. © 2015 Wiley Periodicals, Inc.