PET imaging of rodents is increasingly used in preclinical research, but its utility is limited by spatial resolution and signal-to-noise ratio of the images. A recently developed preclinical PET system uses a clustered-pinhole collimator, enabling high-resolution, simultaneous imaging of PET and SPECT tracers. Pinhole collimation strongly departs from traditional electronic collimation achieved via coincidence detection in PET. We investigated the potential of such a design by direct comparison to a traditional PET scanner. Methods: Two small-animal PET scanners, 1 with electronic collimation and 1 with physical collimation using clustered pinholes, were used to acquire data from Jaszczak (hot rod) and uniform phantoms. Mouse brain imaging using 18 F-FDG PET was performed on each system and compared with quantitative ex vivo autoradiography as a gold standard. Bone imaging using 18 F-NaF allowed comparison of imaging in the mouse body. Images were visually and quantitatively compared using measures of contrast and noise. Results: Pinhole PET resolved the smallest rods (diameter, 0.85 mm) in the Jaszczak phantom, whereas the coincidence system resolved 1.1-mm-diameter rods. Contrast-to-noise ratios were better for pinhole PET when imaging small rods (,1.1 mm) for a wide range of activity levels, but this reversed for larger rods. Image uniformity on the coincidence system (,3%) was superior to that on the pinhole system (5%). The high 18 F-FDG uptake in the striatum of the mouse brain was fully resolved using the pinhole system, with contrast to nearby regions equaling that from autoradiography; a lower contrast was found using the coincidence PET system. For shortduration images (low-count), the coincidence system was superior. Conclusion: In the cases for which small regions need to be resolved in scans with reasonably high activity or reasonably long scan times, a first-generation clustered-pinhole system can provide image quality in terms of resolution, contrast, and the contrast-to-noise ratio superior to a traditional PET system. Smal l-animal PET is an increasingly important tool in biomedical research. A recent development in scanner technology has been the introduction of a focused, clustered-pinhole collimator that enables simultaneous high-spatial-resolution PET and SPECT imaging. Physical collimation of the 511-keV photons produced by positron-electron annihilation, using clustered pinholes, is a substantial change in scanner design as compared with electronic collimation via coincidence detection. Clustered pinholes can offer improved spatial resolution (1) in rodent images, despite a low fraction of single 511-keV photons being detected as compared with the fraction of detected photon pairs in traditional PET. Such scanners are likely suited to different applications. The commonly encountered sensitivity-resolution trade-off has been investigated previously: it has been known for several decades that an improvement in image quality can be achieved via a gain in spatial resolution, even if this gain...