Blood flow imaging is an important tool in cerebrovascular research. Mice are of special interest because of the potential of genetic engineering. Magnetic resonance imaging (MRI) provides three-dimensional noninvasive quantitative methods of cerebral blood flow (CBF) imaging, but these MRI techniques have not yet been validated for mice. The authors compared CBF imaging using flow sensitive alternating inversion recovery (FAIR)-MRI and 14C-Iodoantipyrine (IAP)-autoradiography in a mouse model of acute stroke. Twenty-nine male 129S6/SvEv mice were subjected to filamentous left middle cerebral artery occlusion (MCAo). CBF imaging was performed with 14C-IAP autoradiography and FAIR-MRI using two different anesthesia protocols, namely intravenous infusion of etomidate or inhalation of isoflurane, which differentially affect perfusion. Using 14C-IAP autoradiography, the average CBF in ml/(100 g*min) was 160 ± 34 (isoflurane, n = 5) vs. and 59 ± 21 (etomidate, n = 7) in the intact hemisphere and 43 ± 12 (isoflurane, n = 5) vs. 36 ± 12 (etomidate, n = 7) in the MCAo hemisphere. Using FAIR-MRI, the corresponding average CBFs were 208 ± 56 (isoflurane, intact hemisphere, n = 7), 84 ± 9 (etomidate, intact hemisphere, n = 7), 72 ± 22 (isoflurane, MCAo hemisphere, n = 7) and 48 ± 13 (etomidate, MCAo hemisphere, n = 7). Regression analysis showed a strong linear correlation between CBF measured with FAIR-MRI and 14C-IAP autoradiography, and FAIR-MRI overestimated CBF compared to autoradiography. FAIR-MRI provides repetitive quantitative measurements of hemispheric CBF in a mouse model of stroke.