DESCRIPTION: Rapid sequencing methods have identified functional RNA sequences across all domains of life. Determining the roles of these RNA sequences and their mechanisms of action is central to biology and human health. RNA secondary structure prediction is one of the tools that is commonly used to aid in understanding RNA function, and we have addressed the need for RNA secondary structure prediction by developing RNAstructure. RNAstructure is a user-friendly software package for RNA secondary structure prediction, display, and analysis. It includes methods for structure prediction of a single sequence, including pseudoknots, structure prediction for bimolecular interactions, and prediction of the conserved structure for multiple homologous sequences. Thermodynamic parameters are provided for both RNA and DNA sequences, which extends the structure predictions to DNA. The programs are available with a graphical user interface (for Windows, Mac OS X, or Linux), command line interfaces, and also as web servers. The algorithms are also available for use in other programs as a set of well-documented C++ classes. The package is fully open source, under the GNU Public License. This proposal is for continued development and maintenance of the software. For the next period of support, we are proposing high-impact aims that will keep RNAstructure cutting-edge and user-friendly. We will develop a new interface for facilitating comparative sequence analysis to determine a conserved RNA structure, continue to support and extend the existing programs and interfaces, and develop a new method for structure prediction for pseudoknotted sequences.