Today the majority of clinical molecular imaging procedures are carried out with single-photon emitters and gamma cameras, in planar mode and single-photon emission computed tomography (SPECT) mode. Thanks to the development of advanced multi-pinhole collimation technologies, SPECT imaging of small experimental animals is rapidly gaining in popularity. Whereas resolutions in routine clinical SPECT are typically larger than 1 cm (corresponding to >1,000 microl), it has recently proved possible to obtain spatial resolutions of about 0.35 mm ( approximately 0.04 microl) in the mouse. Meanwhile, SPECT systems that promise an even better performance are under construction. The new systems are able to monitor functions in even smaller structures of the mouse than was possible with dedicated small animal positron emission tomography ( approximately 1 mm resolution, corresponding to 1 microl). This paper provides a brief history of image formation with pinholes and explains the principles of pinhole imaging and pinhole tomography and the basics of modern image reconstruction methods required for such systems. Some recently introduced ultra-high-resolution small animal SPECT instruments are discussed and new avenues for improving system performance are explored. This may lead to many completely new biomedical applications. We also demonstrate that clinical SPECT systems with focussing pinhole gamma cameras will be able to produce images with a resolution that may become superior to that of PET for major clinical applications. A design study of a cardiac pinhole SPECT system indicates that the heart can be imaged an order of magnitude faster or with much more detail than is possible with currently used parallel-hole SPECT (e.g. 3-4 mm instead of approximately 8 mm system resolution).