We have developed a pinhole collimator for small animal imaging using dual-isotopes, such as gamma and positron emitters. A lead cylinder containing a pinhole was placed around the subject (a small animal). The cylinder was equipped with a non-collimator gamma camera, and dual-isotope (99mTc-MIBI and 18F-FDG) SPECT was performed on a Wistar King Aptekman/hok (WKAH) rat. System planar sensitivity and Full-Width at Half-Maximum (FWHM) were measured for each radionuclide. System planar sensitivities for 99mTc and 18F SPECT were 2 and 7 cps/MBq, respectively. FWHMs for 99mTc and 18F SPECT were 2.0±0.5 and 2.7±0.5 mm, respectively. The collimator is relatively light (23 kg), and thus SPECT projection data could be acquired by rotating the gamma camera while the object remained stationary. The pinhole collimator can be used with a conventional rotating gamma camera. The present study demonstrated that it is possible to image organs in vivo in sufficient detail using the newly developed pinhole collimator. Further refinements to the experimental procedure may provide simultaneous high-resolution imaging of small animals using positron and gamma emitters with this collimator.