AbstractMotivation: Understanding the architecture and function of RNA molecules requires methods for comparing and analyzing their 3D structures. Although a structural alignment of short RNAs is achievable in a reasonable amount of time, large structures represent much bigger challenge. However, the growth of the number of large RNAs deposited in the PDB database calls for the development of fast and accurate methods for analyzing their structures, as well as for rapid similarity searches in databases.Results: In this article a novel algorithm for an RNA structural comparison SETTER (SEcondary sTructure-based TERtiary Structure Similarity Algorithm) is introduced. SETTER uses a pairwise comparison method based on 3D similarity of the so-called generalized secondary structure units. For each pair of structures, SETTER produces a distance score and an indication of its statistical significance. SETTER can be used both for the structural alignments of structures that are already known to be homologous, as well as for 3D structure similarity searches and functional annotation. The algorithm presented is both accurate and fast and does not impose limits on the size of aligned RNA structures.Availability: The SETTER program, as well as all datasets, is freely available from http://siret.cz/hoksza/projects/setter/.Contact: hoksza@ksi.mff.cuni.cz, or svozild@vscht.czSupplementary information: Supplementary data are available at Bioinformatics online.