The objective of this investigation was to evaluate small-field-of-view, ultra-high-resolution pinhole collimation for a rotating-camera SPECT system that could be used to image small laboratory animals. Pinhole collimation offers distinct advantages over conventional parallel-hole collimation when used to image small objects. Since geometric sensitivity increases markedly for points close to the pinhole, small-diameter and high-magnification pinhole geometries may be useful for selected imaging tasks when used with large-field-of-view scintillation cameras. The use of large magnifications can minimize the loss of system resolution caused by the intrinsic resolution of the scintillation camera. A pinhole collimator has been designed and built that can be mounted on one of the scintillation cameras of a triple-head SPECT system. Three pinhole inserts with approximate aperture diameters of 0.6, 1.2 and 2.0 mm have been built and can be mounted individually on the collimator housing. When a ramp filter is used with a three-dimensional (3D) filtered backprojection (FBP) algorithm, the three apertures have in-plane SPECT spatial resolutions (FWHM) at 4 cm of 1.5, 1.9 and 2.8 mm, respectively. In-air point source sensitivities at 4 cm from the apertures are 0.9, 2.6 and 5.7 counts s(-1) microCi(-1) (24, 70 and 154 counts s(-1) MBq(-1)) for the 0.6, 1.2 and 2.0 mm apertures, respectively. In vitro image quality was evaluated with a micro-cold-rod phantom and a micro-Defrise phantom using both the 3D FBP algorithm and a 3D maximum likelihood-expectation maximization (ML-EM) algorithm. In vivo image quality was evaluated using two (315 and 325 g) rats. Ultra-high-resolution pinhole SPECT is an inexpensive and simple approach for imaging small animals that can be used with existing rotating-camera SPECT system.