Micro positron emission tomography (PET) and micro single-photon emission computed tomography (SPECT), used for imaging small animals, have become essential tools in developing new pharmaceuticals and can be used, among other things, to test new therapeutic approaches in animal models of human disease, as well as to image gene expression. These imaging techniques can be used noninvasively in both detection and quantification. However, functional images provide little information on the structure of tissues and organs, which makes the localization of lesions difficult. Image fusion techniques can be exploited to map the functional images to structural images, such as X-ray computed tomography (CT), to support target identification and to facilitate the interpretation of PET or SPECT studies. Furthermore, the mapping of two functional images of SPECT and PET on a structural CT image can be beneficial for those in vivo studies that require two biological processes to be monitored simultaneously. This paper proposes an automated method for registering PET, CT, and SPECT images for small animals. A calibration phantom and a holder were used to determine the relationship among three-dimensional fields of view of various modalities. The holder was arranged in fixed positions on the couches of the scanners, and the spatial transformation matrix between the modalities was held unchanged. As long as objects were scanned together with the holder, the predetermined matrix could register the acquired tomograms from different modalities, independently of the imaged objects. In this work, the PET scan was performed by Concorde's microPET R4 scanner, and the SPECT and CT data were obtained using the Gamma Medica's X-SPECT/CT system. Fusion studies on phantoms and animals have been successfully performed using this method. For microPET-CT fusion, the maximum registration errors were 0.21 mm ± 0.14 mm, 0.26 mm ± 0.14 mm, and 0.45 mm ± 0.34 mm in the X (right-left), Y (upper lower), and Z (rostral-caudal) directions, respectively; for the microPET-SPECT fusion, they were 0.24 mm ± 0.14 mm, 0.28 mm ± 0.15 mm, and 0.54 mm ± 0.35 mm in the X, Y, and Z directions, respectively. The results indicate that thi- s simple method can be used in routine fusion studies.