In order to estimate free radical reactions and image them in the brain of living animals, a nitroxyl spin-probe, carboxy-PROXYL acetoxymethyl ester (CxP-AM) was newly synthesized. CxP-AM was designed to be hydrolyzed by esterase, but not by lipase, so that it would pass through the blood-brain barrier and be retained in the cytosolic phase of parenchymal cells in the brain after intravenous injection. The pharmacokinetics of CxP-AM was compared with those of carboxy-PROXYL (CxP) and its methyl ester (CxP-M). Carboxyl esterase almost completely hydrolyzed CxP-AM within 3 min. After intravenous injection, the brain retained 1.8 times more CxP-AM than CxP-M, and retained it for more than 30 min. Electron spin resonance computed tomographic (ESR-CT) imaging of CxP-AM in the heads of mice produced marked contrast in the encephalon region, while CxP was distributed only in the extracranial region and CxP-M was distributed in both regions, confirming the pharmacokinetics of CxP-AM. The decay rate of CxP-AM determined with time-resolved ESR-CT imaging was different in the two brain regions, suggesting regional differences in the total reducing capability. CxP-AM should become a powerful probe for the investigation and diagnosis of free radical reactions and their imaging in the brain.