Three-dimensional motif search is becoming increasingly important both in the search for molecular signatures within a tomographic reconstruction, at low resolution, and in the search for atomic structures within high-resolution cryo-EM maps of macromolecular complexes. The present work describes the implementation of a fast local correlation algorithm suitable for template matching in the SPIDER environment. Two examples are given, one in each of the areas of application: (i) within a 7.8 Angstrom single-particle reconstruction of the Escherichia coli ribosome, four proteins and one RNA structure were located with high accuracy; (ii) within a cryo-tomogram of sarcoplasmic reticulum vesicles, ryanodine receptors were located in positions that agreed with expert knowledge. (C) 2003 Elsevier Inc. All rights reserved.