An (15)O-labeled water (H(2)(15)O) steady-state method for quantitative measurement of cerebral blood flow (CBF), which is less stressful to small animals with a few point blood sampling, was developed. After a simulation using a dose meter to achieve stable H(2)(15)O radioactivity in the blood with a multiprogramming syringe pump programmed for slowly increasing injection volume, 10 rats were studied with the injection method. Arterial blood was sampled every minute during 6-minute positron emission tomography (PET) scans. After the PET scan, N-isopropyl-p-[(125)I]-iodoamphetamine ((125)I-IMP) was injected into the same rat to measure CBF using the autoradiography method based on a microsphere model. Regions of interest were placed on the whole brain in H(2)(15)O-PET and (125)I-IMP-autoradiography images, and CBF values calculated from both methods were compared. Radioactivity in the dose meter achieved equilibrium ∼1 minute after starting the H(2)(15)O injection. In rat studies, radioactivity in the blood and brain rapidly achieved equilibrium at 2 minutes after administration. The correlation of CBF values of H(2)(15)O PET (49.2±5.4 mL per 100 g per minute) and those of (125)I-IMP autoradiography (49.1±5.2 mL per 100 g per minute) was excellent (y=1.01x-0.37, r(2)=0.97). The H(2)(15)O steady-state method with a continuously increasing injection is useful for CBF measurement in small animal studies, especially when multiple scans are required in the same animal.