1-Acetoxy-3-carbamoyl-2,2,5,5-tetramethylpyrrolidine (ACP) is a unique probe for in vivo measurements of reactive oxygen species (ROS), because it is hydrolyzed by esterase to a hydroxylamine form (CP-H), which is oxidized to an electron spin resonance-detectable nitroxyl radical (CP) by a reaction with superoxide anion radical, etc. Although a knowledge of pharmacokinetics is essential for the use of ACP in vivo, such information is limited. We investigated the pharmacokinetics of ACP in mice by examining the time course of the tissue distribution of ACP, CP-H, and CP after intravenous or intraperitoneal injection of ACP. Esterase activity for ACP in tissue homogenates was also measured. The concentration of ACP decreased in all tissues obeying a one-compartment model. ACP was hydrolyzed to CP-H in the liver and kidney predominantly, and the first-pass effect of liver on the hydrolysis of ACP was very large. A homogeneous biodistribution of CP-H was obtained 10 min after the injection of ACP regardless of the injection route, and concentrations remained stable over at least 20 min. Because of these pharmacokinetic properties, ACP should be suitable for the imaging of ROS in animals.