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PET and SPECT have been widely used to investigate the physiological function of animals in vivo. However, little efforts have been done to estimate absolute physiological parameters, i.e. blood flow of small animals. The present study was aimed at the absolute quantitation of myocardial blood flow of rats by means of the dynamic SPECT fitted with a pinhole collimator with a careful determination of the arterial input function (IF). The center-of-rotation was carefully aligned to the center of the field-of-view of a fixed gamma camera with the accuracy < 0.05 mm. A rat was placed on a rotating device that fixes the rat in a stand position. The arterial blood samples were frequently collected and their radioactivity concentration was measured using a well counter cross-calibrated to the SPECT images. Dynamic SPECT (the step-and-shoot mode) was initiated at 5 min after the injection of 201TlCl into the tail vein. Acquisition period was 10 sec at each rotation angle, and 120 view projection data were obtained. The 360-degree complete data sets were obtained at approximately 20 min interval for 5 time frames. Images were reconstructed by filtered-back projection technique with Feldkamp algorithm. The cross-calibration factor was determined using a cylindrical phantom of 5 cm diameter filled with the 201TlCl solution. Regions-of-interest were placed on the left ventricular wall to generate the tissue time activity curve (TTAC). TTAC and IF were fitted to the previously validated single-tissue compartment model to estimate the regional myocardial blood flow (rMBF) and volume of distribution (Vd) of thallium. The present system provided clear images of myocardial uptake of 201TlCl, and the time-dependent change of the tissue radioactivity concentration in regional basis, which was statistically sufficient for applying the compartment model analysis. The kinetic analysis yielded the rMBF of approximately 0.77 ml/min/g, which appeared to be an acceptable value with a consideration of contribution of partial volume effect and other error sources. Vd of approximately 91.9 ml/ml was also consistent with the know value of the potassium potential across the cell membrane. These results strongly suggested the potential of the dynamic pinhole SPECT as a tool for absolute qua- ntitation of physiological parameters in small animals.