The purpose of this study was to evaluate the accuracy of myocardial imaging by means of high-resolution single-photon emission tomography (SPET) with a pinhole collimator in rats with experimental infarction. Myocardial infarctions were induced in male Wistar rats by ligation of the left coronary artery for 30 min, followed by reperfusion. Two days after the reperfusion, pinhole SPET was performed after the intravenous administration of 111 MBq of thallium-201 chloride, using a rotating gamma camera equipped with a pinhole insert (2.0-mm aperture) in a low-energy pinhole collimator. SPET projection data were collected at 6 degrees increments over 360 degrees using a 4-cm radius of rotation to reconstruct the short- and long-axis images. Projection data were acquired in 15 or 30 s, the SPET imaging being accomplished within 40 min after the injection of 201Tl. After SPET, the rats were sacrificed to remove the hearts for autoradiography (ARG) and nitroblue tetrazolium (NBT) staining as a visual correlative study. Quantitative correlative studies between pinhole SPET and ARG were performed with linear regression analysis for infarct size and distribution properties (relative counts on SPET images and relative density on autoradiographs) on the short-axis sections. All infarcts (4 mm in minimum diameter) in seven rats were detected by pinhole SPET. The SPET images in rats with or without myocardial infarction were consistent with the findings of ARG and NBT staining. There were significant correlations between pinhole SPET and ARG with respect to the infarct size (r=0.933, P <0.001; n=15) and the relative radiotracer distribution (r=0.931, P <0.001; n=68). This study therefore confirmed the accuracy of myocardial pinhole SPET imaging in rats with myocardial infarction. This method may partially substitute for ARG and prove useful for assessing new myocardial imaging agents in vivo in small laboratory animals.